Ultrasound contrast agents and process for the preparation thereof

ABSTRACT

Injectable aqueous suspension of microbubbles filled with a biocompatible gas and a method of preparation thereof. At least 10% of the total volume of gas contained in the microbubbles is contained in microbububbles with a diameter of 1.5 μm or less. The microbubbles can be obtained by preparing an emulsion comprising an aqueous medium, a phospholipid and a water immiscible organic solvent. The emulsion is then freeze-dried and then reconstituted in an aqueous suspension of gas-filled microbubbles.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of co-pending U.S.application, U.S. Ser. No. ______, filed Aug. 2, 2005, which is anational stage application of international applicationPCT/IB2004/000243, filed Feb. 3, 2004, which claims priority to and thebenefit of European application EP03002375.8, filed Feb. 4, 2003, all ofwhich are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a process for the preparation of a dryor lyophilized formulation useful for preparing a gas containingcontrast agent usable in diagnostic imaging and to a process forpreparing said gas containing contrast agent.

The invention also includes dry formulations prepared by this process,which may be reconstituted to form contrast agent suspensions useful indiagnostic imaging. The invention further includes suspensions of gasfilled microbubbles useful in diagnostic imaging prepared using dryformulations of the invention as well as containers or two componentkits containing the dry formulations of the invention.

BACKGROUND OF THE INVENTION

Rapid development of ultrasound contrast agents in the recent years hasgenerated a number of different formulations, which are useful inultrasound imaging of organs and tissue of human or animal body. Theseagents are designed to be used primarily as intravenous orintra-arterial injectables in conjunction with the use of medicalechographic equipment which employs for example, B-mode image formation(based on the spatial distribution of backscatter tissue properties) orDoppler signal processing (based on Continuous Wave or pulsed Dopplerprocessing of ultrasonic echoes to determine blood or liquid flowparameters).

A class of injectable formulations useful as ultrasound contrast agentsincludes suspensions of gas bubbles having a diameter of few micronsdispersed in an aqueous medium.

Use of suspensions of gas bubbles in carrier liquid, as efficientultrasound reflectors is well known in the art. The development ofmicrobubble suspensions as echopharmaceuticals for enhancement ofultrasound imaging followed early observations that rapid intravenousinjections of aqueous solutions can cause dissolved gases to come out ofsolution by forming bubbles. Due to their substantial difference inacoustic impedance relative to blood, these intravascular gas bubbleswere found to be excellent reflectors of ultrasound. The injection ofsuspensions of gas bubbles in a carrier liquid into the blood stream ofa living organism strongly reinforces ultrasonic echography imaging,thus enhancing the visualisation of internal organs. Since imaging oforgans and deep seated tissues can be crucial in establishing medicaldiagnosis, a lot of effort has been devoted to the development of stablesuspensions of highly concentrated gas bubbles which at the same timewould be simple to prepare and administer, would contain a minimum ofinactive species and would be capable of long storage and simpledistribution.

The simple dispersion of free gas bubbles in the aqueous medium ishowever of limited practical interest, since these bubbles are ingeneral not stable enough to be useful as ultrasound contrast agents.

Interest has accordingly been shown in methods of stabilising gasbubbles for echography and other ultrasonic studies, for example usingemulsifiers, oils, thickeners or sugars, or by entrapping orencapsulating the gas or a precursor thereof in a variety of systems.These stabilized gas bubbles are generally referred to in the art as“microvesicles”, and may be divided into two main categories.

A first category of stabilized bubbles or microvesicles is generallyreferred to in the art as “microbubbles” and includes aqueoussuspensions in which the bubbles of gas are bounded at the gas/liquidinterface by a very thin envelope involving a surfactant (i.e. anamphiphilic material) disposed at the gas to liquid interface. A secondcategory of microvesicles is generally referred to in the art as“microballoons” or “microcapsules” and includes suspensions in which thebubbles of gas are surrounded by a solid material envelope formed ofnatural or synthetic polymers. Examples of microballoons and of thepreparation thereof are disclosed, for instance, in European patentapplication EP 0458745. Another kind of ultrasound contrast agentincludes suspensions of porous microparticles of polymers or othersolids, which carry gas bubbles entrapped within the pores of themicroparticles. The present invention is particularly concerned withcontrast agents for diagnostic imaging including an aqueous suspensionof gas microbubbles, i.e. microvesicles which are stabilized essentiallyby a layer of amphiphilic material.

Microbubble suspensions are typically prepared by contacting powderedamphiphilic materials, e.g. freeze-dried preformed liposomes orfreeze-dried or spray-dried phospholipid suspensions, with air or othergas and then with aqueous carrier, agitating to generate a microbubblesuspension which must then be administered shortly after itspreparation.

Examples of aqueous suspensions of gas microbubbles and preparationthereof can be found for instance in U.S. Pat. No. 5,271,928, U.S. Pat.No. 5,445,813, U.S. Pat. No. 5,413,774, U.S. Pat. Nos. 5,556,610,5,597,549, U.S. Pat. No. 5,827,504.

WO97/29783 discloses an alternative process for preparing gasmicrobubble suspensions, comprising generating a gas microbubbledispersion in an appropriate phospholipid-containing aqueous medium andthereafter subjecting the dispersion to lyophilisation to yield a driedreconstitutable product. The so prepared dried products arereconstitutable in aqueous media requiring only minimal agitation. Asmentioned in said document, the size of the so generated microbubbles isconsistently reproducible and in practice is independent from the amountof agitation energy applied during reconstitution, being determined bythe size of the microbubbles formed in the initial microbubbledispersion. The Applicant has however observed that the amount ofagitation energy applied for generating the gas microbubble dispersionin the phospholipid-containing aqueous medium may be excessively high,particularly when small diameter microbubbles are to be obtained (e.g.23000 rpm for 10 minutes, for obtaining a dispersion of bubbles having avolume mean diameter of about 3 μm). This high agitation energy maydetermine local overheating in the aqueous dispersion of microbubbles,which may in turn cause degradation of the phospholipids contained inthe aqueous medium. In addition, the effects of an excessively highagitation energy are in general difficult to control and may result inan uncontrollable size distribution of the final microbubbles.Furthermore, this process involves a continuous flow of gas into theaqueous medium during the generation of microbubbles, thus requiring theuse of relevant amounts of gases.

WO 94/01140 discloses a further process for preparing microvesiclesuspensions reconstitutable in an aqueous medium, which compriseslyophilizing aqueous emulsions containing parenterally acceptableemulsifiers, non polar liquids and lipid-soluble or water-insoluble“structure-builders”. Poloxamers and phospholipids are mentioned asparenterally acceptable emulsifiers, while mixtures of these two areemployed in the working examples. Cholesterol is the preferredwater-insoluble structure-builder, which is employed in the workingexamples. The lyophilized product is then reconstituted in water, togive aqueous suspension of gas-filled microvesicles. The gas-filledmicrovesicles resulting from the reconstitution step are thus defined byan envelope of different materials, including emulsifiers such aspoloxamers and water-insoluble structure-builders such as cholesterol.

The process is said to result into an emulsion with particles' sizelower than 4 μm, preferably lower than 2 μm, down to 0.5 μm. TheApplicant has however noticed that while the reconstitution step mayfinally result in microvesicles having a numerical mean diameter of lessthan 2 μm, the corresponding size distribution of the microvesiclespopulation is nevertheless relatively broad. In addition, the conversionstep from the emulsion microparticles, obtained according to the aboveprocess, into gas microbubbles results in rather low yield.

The Applicant has now found that a much narrower distribution ofmicrobubbles size can be obtained if a phospholipid is used as the mainemulsifier of the above emulsion and if the above process is conductedin the substantial absence of the above water-insolublestructure-builders. In addition, the substantial absence of saidwater-insoluble structure-builders allows to substantially increase theconversion yield from emulsion microparticles into gas microbubbles. TheApplicant has further observed that the above process may result in afurther narrower size distribution of microbubbles and in an increasedyield if the phospholipid is essentially the only emulsifier present inthe emulsion.

The Applicant has also found that by applying a rather low agitationenergy to an aqueous-organic emulsion during the process as abovespecified, it is possible to obtain microbubbles having a very smalldiameter and reduced size distribution.

SUMMARY OF THE INVENTION

An aspect of the invention relates to an injectable contrast agentcomprising gas-filled microbubbles stabilized by a stabilizing layerpredominantly comprising a phospholipid in an aqueous carrier liquid,wherein at least 10% of the total volume of gas contained in themicrobubbles is contained in microbubbles with a diameter of 1.5 μm orless. Preferably, at least 25%, more preferably at least 50% and evenmore preferably at least 70% of the total volume of gas is contained inmicrobubbles with a diameter of less than 1.5 μm.

According to a preferred embodiment, said gas-filled microbubbles have avolume median diameter (D_(V50)) and a number mean diameter (D_(N)) suchthat the D_(V50)/D_(N) ratio is of about 2.00 or lower, more preferablyof about 1.60 or lower and even more preferably of about 1.30 or lower.

Preferably, said injectable contrast agent is in the form of an aqueoussuspension of gas-filled microbubbles. According to a preferredembodiment, said aqueous suspension is obtained by a method whichcomprises the steps of:

-   -   preparing an aqueous-organic emulsion comprising i) an aqueous        medium including water, ii) an organic solvent substantially        immiscible with water, iii) an emulsifying composition of        amphiphilic materials comprising more than 50% by weight of a        phospholipid and iv) a lyoprotecting agent;    -   lyophilizing said emulsified mixture, to obtain a lyophilized        matrix comprising said phospholipid;    -   contacting said lyophilized matrix with a biocompatible gas; and    -   reconstituting said lyophilized matrix by dissolving it in a        physiologically acceptable aqueous carrier liquid.

A further aspect of the invention relates to a method for preparing alyophilized matrix which, upon contact with an aqueous carrier liquidand a gas, is reconstitutable into a suspension of gas-filledmicrobubbles stabilized predominantly by a phospholipid, said methodcomprising the steps of:

-   -   preparing an aqueous-organic emulsion comprising i) an aqueous        medium including water, ii) an organic solvent substantially        immiscible with water comprising, dispersed therein, an        emulsifying composition of amphiphilic materials comprising more        than 50% by weight of a phospholipid and iv) a lyoprotecting        agent;    -   lyophilizing said emulsified mixture, to obtain a lyophilized        matrix comprising said phospholipid.

DETAILED DESCRIPTION OF THE INVENTION

The injectable contrast agent of the present invention can be obtainedfrom a lyophilized matrix of a reconstitutable suspension of gas-filledmicrobubbles predominantly stabilized by a phospholipid, said matrixbeing obtained from a method which comprises preparing anaqueous-organic emulsion comprising i) an aqueous medium, ii) an organicsolvent substantially immiscible with water; iii) a phospholipid and iv)a lyoprotecting agent, and subsequently lyophilizing said emulsion.

The aqueous medium is preferably a physiologically acceptable carrier.The term “physiologically acceptable” includes to any compound, materialor formulation which can be administered, in a selected amount, to apatient without negatively affecting or substantially modifying itsorganism's healthy or normal functioning (e.g. without determining anystatus of unacceptable toxicity, causing any extreme or uncontrollableallergenic response or determining any abnormal pathological conditionor disease status).

Suitable aqueous liquid carriers are water, typically sterile, pyrogenfree water (to prevent as much as possible contamination in theintermediate lyophilized product), aqueous solutions such as saline(which may advantageously be balanced so that the final product forinjection is not hypotonic), or aqueous solutions of one or moretonicity adjusting substances such as salts or sugars, sugar alcohols,glycols or other non-ionic polyol materials (eg. glucose, sucrose,sorbitol, mannitol, glycerol, polyethylene glycols, propylene glycolsand the like).

The Organic Solvent

As used herein the term “substantially immiscible with water” referredto the organic solvent means that, when said solvent is admixed withwater, two separate phases are formed. Water immiscible solvent aregenerally also known in the art as a polar or non-polar solvents, asopposed to polar solvents (such as water). Water immiscible solvents arein general substantially insoluble in water. For the purposes of thepresent invention, organic solvents suitable for being emulsified withthe aqueous solvent are typically those solvents having a solubility inwater of less than about 10 g/l. Preferably, the solubility of saidsolvent in water is of about 1.0 g/l or lower, more preferably about 0.2g/l or lower and much more preferably about 0.01 g/l or lower.Particularly preferred solvents are those having a solubility in waterof 0.001 g/l or lower. Particularly insoluble organic solvents (e.g.perfluorocarbons) may have a solubility down to about 1.0·10⁻⁶ g/l (e.g.perfluorooctane, 1.66·10⁻⁶ g/l).

The organic solvent is preferably lyophilisable, i.e. said solvent has asufficiently high vapour pressure at the lyophilization temperatures,e.g. between −30° C. and 0° C., to allow for an effective and completeevaporation/sublimation within acceptable times, e.g. 24-48 hours.Preferably, the vapour pressure of the organic solvent is higher thanabout 0.2 kPa at 25° C.

The organic solvent can be selected from a broad range of solvents andany chemical entity that is water-immiscible and lyophilisable, asindicated above, and being preferably liquid at room temperature (25°C.). If a solvent having a boiling point lower than room temperature isused, the vessel containing the emulsifying mixture can advantageouslybe cooled below the boiling point of said solvent, e.g. down to 5° C. or0° C. As said solvent will be completely removed during thelyophilization step, no particular constraints exist except that itshould not contain contaminants that cannot be removed throughlyophilisation or that are not acceptable for use in an injectablecomposition.

Suitable organic solvents include but are not limited to alkanes, suchas branched or, preferably, linear (C₅-C₁₀) alkanes, e.g. pentane,hexane, heptane, octane, nonane, decane; alkenes, such as (C₅-C₁₀)alkenes, e.g. 1-pentene, 2-pentene, 1-octene; cyclo-alkanes, such as(C₅-C₈)-cycloalkanes optionally substituted with one or two methylgroups, e.g. cyclopentane, cyclohexane, cyclooctane,1-methyl-cyclohexane; aromatic hydrocarbons, such as benzene and benzenederivatives substituted by one or two methyl or ethyl groups, e.g.benzene, toluene, ethylbenzene, 1,2-dimethylbenzene,1,3-dimethylbenzene; alkyl ethers and ketones such as di-butyl ether anddi-isopropylketone; halogenated hydrocarbons or ethers, such aschloroform, carbon tetrachloride,2-chloro-1-(difluoromethoxy)-1,1,2-trifluoroethane (enflurane),2-chloro-2-(difluoromethoxy)-1,1,1-trifluoroethane (isoflurane),tetrachloro-1,1-difluoroethane, and particularly perfluorinatedhydrocarbons or ethers, such as perfluoropentane, perfluorohexane,perfluoroheptane, perfluoromethylcyclohexane, perfluorooctane,perfluorononane, perfluorobenzene and perfluorodecalin,methylperfluorobutylether, methylperfluoroisobutylether,ethylperfluorobutylether, ethylperfluoroisobutylether; and mixturesthereof.

The amount of solvent is generally comprised from about 1% to about 50%by volume with respect to the amount of water used for the emulsion.Preferably said amount is from about 1% to about 20%, more preferablyfrom about 2% to about 15% and even more preferably from about 5% toabout 10%. If desired, a mixture of two or more of the above listedorganic solvents can be used, the overall amount of organic solvent inthe emulsifying mixture being within the above range.

Lyoprotective Agent

The term lyoprotective agent or “lyoprotectant” refers to a compoundwhich, when included in a formulation to be lyophilized, will protectthe chemical compounds from the deleterious effects of freezing andvacuumizing, such as those usually accompanying lyophilization, e.g.damage, adsorption and loss from vacuum utilized in lyophilization. Inaddition, after the lyophilization step, said lyoprotective agentpreferably results in a solid matrix (“bulk”) which supports thelyophilized phospholipid.

The present invention is not limited to the use of a specificlyoprotectant, and examples of suitable lyoprotectants include, but arenot limited to, carbohydrates such as the saccharides, mono-, di- orpoly-saccharides, e.g. glucose, galactose, fructose, sucrose, trehalose,maltose, lactose, amylose, amylopectin, cyclodextrins, dextran, inuline,soluble starch, hydroxyethyl starch (HES), sugar alcohols e.g. mannitol,sorbitol and polyglycols such as polyethyleneglycols. A substantial listof agents with lyoprotective effects is given in Acta Pharm. Technol.34(3), pp. 129-139 (1988), the content of which is incorporated hereinby reference. Said lyoprotective agents can be used singularly or asmixtures of one or more compounds.

Preferred lyoprotectants include mannitol and polysaccharides such asdextrans (in particular those with molecular weights above 1500daltons), inulin, soluble starch, hydroxyethyl starch andpolyethyleneglycols, preferably of MW from about 1000 to about 30000daltons, more preferably from 2000 to 8000 daltons (e.g. PEG4000).

Mixtures of mannitol or polysaccharides such as dextrans, inulin,soluble starch, hydroxyethyl starch with saccharides such as glucose,maltose, lactose, sucrose, trehalose and erythritol also provideexcellent results.

Likewise, the present invention is not limited to any particular amountof lyoprotectant used. However the optimal weight concentration oflyoprotective agents in the emulsion prior to the lyophilisation iscomprised between about 1 and about 25%, preferably between about 2 andabout 20%, and even more preferably between about 5 and about 10%.

A higher amount can be employed if it is also necessary to provide adesired “bulk” to the lyophilized product.

The lyoprotective agent is preferably added to the aqueous-organicmixture before emulsification of the same and in this case theemulsification of the aqueous-organic mixture is thus carried out in thepresence of the lyoprotective agents. Alternatively, the lyoprotectantcan be added to the aqueous-organic mixture after the emulsificationthereof. In the first case, the lyoprotectant is preferably added to theaqueous medium, before admixing it with the organic solvent. If desired,it is also possible to combine the two, e.g. by adding part of thelyoprotective agent to the aqueous phase used for the preparation of theemulsion and part to the thus obtained emulsion. If desired, alsocryoprotective agents, such as glycerol, can further be added to theemulsion for protecting the chemical compounds from the deleteriouseffects of freezing.

Phospholipids

According to the present description and claims, the term phospholipidis intended to encompass any amphiphilic phospholipidic compound themolecules of which are capable of forming a film of material (typicallyin the form of a mono-molecular layer) at the gas-water boundaryinterface in the final microbubbles suspension. Accordingly, thesematerial are also referred to in the art as “film-formingphospholipids”. Similarly, in the emulsified mixture, these amphiphiliccompounds are typically disposed at the interface between the aqueousmedium and the organic solvent substantially insoluble in water, thusstabilizing the emulsified solvent microdroplets. The film formed bythese compounds at the gas-water or water-solvent interface can beeither continuous or discontinuous. In the latter case, thediscontinuities in the film should not however be such as to impair thestability (e.g. pressure resistance, resistance to coalescence, etc.) ofthe suspended microbubbles or of the emulsified microdroplets,respectively.

The term “amphiphilic compound” as used herein includes compounds havinga molecule with a hydrophilic polar head portion (e.g. a polar or ionicgroup), capable of interacting with an aqueous medium, and a hydrophobicorganic tail portion (e.g. a hydrocarbon chain), capable of interactingwith e.g. an organic solvent. These compounds thus generally act as“surface active agent”, i.e. compounds which are capable of stabilizingmixtures of otherwise generally immiscible materials, such as mixturesof two immiscible liquids (e.g. water and oil), mixtures of liquids withgases (e.g. gas microbubbles in water) or mixtures of liquids withinsoluble particles (e.g. metal nanoparticles in water).

Amphiphilic phospholipid compounds typically contain at least onephosphate group and at least one, preferably two, lipophilic long-chainhydrocarbon group.

Examples of suitable phospholipids include esters of glycerol with oneor preferably two (equal or different) residues of fatty acids and withphosphoric acid, wherein the phosphoric acid residue is in turn bound toa hydrophilic group, such as choline (phosphatidylcholines—PC), serine(phosphatidylserines—PS), glycerol (phosphatidylglycerols—PG),ethanolamine (phosphatidylethanolamines—PE), inositol(phosphatidylinositol), and the like groups. Esters of phospholipidswith only one residue of fatty acid are generally referred to in the artas the “lyso” forms of the phospholipid. Fatty acids residues present inthe phospholipids are in general long chain aliphatic acids, typicallycontaining from 12 to 24 carbon atoms, preferably from 14 to 22; thealiphatic chain may contain one or more unsaturations or is preferablycompletely saturated. Examples of suitable fatty acids included in thephospholipids are, for instance, lauric acid, myristic acid, palmiticacid, stearic acid, arachidic acid, behenic acid, oleic acid, linoleicacid, and linolenic acid. Preferably, saturated fatty acids such asmyristic acid, palmitic acid, stearic acid and arachidic acid areemployed.

Further examples of phospholipid are phosphatidic acids, i.e. thediesters of glycerol-phosphoric acid with fatty acids; sphingolipidssuch as sphingomyelins, i.e. those phosphatidylcholine analogs where theresidue of glycerol diester with fatty acids is replaced by a ceramidechain; cardiolipins, i.e. the esters of 1,3-diphosphatidylglycerol witha fatty acid; glycolipids such as gangliosides GM1 (or GM2) orcerebrosides; glucolipids; sulfatides and glycosphingolipids.

As used herein, the term phospholipids include either naturallyoccurring, semisynthetic or synthetically prepared products that can beemployed either singularly or as mixtures.

Examples of naturally occurring phospholipids are natural lecithins(phosphatidylcholine (PC) derivatives) such as, typically, soya bean oregg yolk lecithins.

Examples of semisynthetic phospholipids are the partially or fullyhydrogenated derivatives of the naturally occurring lecithins. Preferredphospholipids are fatty acids di-esters of phosphatidylcholine,ethylphosphatidylcholine, phosphatidylglycerol, phosphatidic acid,phosphatidylethanolamine, phosphatidylserine or of sphingomyelin.

Examples of preferred phospholipids are, for instance,dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidylcholine(DMPC), dipalmitoyl-phosphatidylcholine (DPPC),diarachidoyl-phosphatidylcholine (DAPC), distearoyl-phosphatidylcholine(DSPC), dioleoyl-phosphatidylcholine (DOPC), 1,2Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC),dipentadecanoyl-phosphatidylcholine (DPDPC),1-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC),1-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC),1-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC),1-stearoyl-2-palmitoyl-phosphatidylcholine (SPPC),),1-palmitoyl-2-oleylphosphatidylcholine (POPC),1-oleyl-2-palmitoyl-phosphatidylcholine (OPPC),dilauroyl-phosphatidylglycerol (DLPG) and its alkali metal salts,diarachidoylphosphatidyl-glycerol (DAPG) and its alkali metal salts,dimyristoylphosphatidylglycerol (DMPG) and its alkali metal salts,dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal salts,distearoylphosphatidylglycerol (DSPG) and its alkali metal salts,dioleoyl-phosphatidylglycerol (DOPG) and its alkali metal salts,dimyristoyl phosphatidic acid (DMPA) and its alkali metal salts,dipalmitoyl phosphatidic acid (DPPA) and its alkali metal salts,distearoyl phosphatidic acid (DSPA), diarachidoylphosphatidic acid(DAPA) and its alkali metal salts, dimyristoyl-phosphatidylethanolamine(DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidyl-ethanolamine (DSPE), dioleylphosphatidyl-ethanolamine(DOPE), diarachidoylphosphatidylethanolamine (DAPE),dilinoleylphosphatidylethanolamine (DLPE), dimyristoylphosphatidylserine (DMPS), diarachidoyl phosphatidylserine (DAPS),dipalmitoyl phosphatidylserine (DPPS), distearoylphosphatidylserine(DSPS), dioleoylphosphatidylserine (DOPS), dipalmitoyl sphingomyelin(DPSP), and distearoylsphingomyelin (DSSP).

The term phospholipid further includes modified phospholipid, e.g.phospholipids where the hydrophilic group is in turn bound to anotherhydrophilic group. Examples of modified phospholipids arephosphatidylethanolamines modified with polyethylenglycol (PEG), i.e.phosphatidylethanolamines where the hydrophilic ethanolamine moiety islinked to a PEG molecule of variable molecular weight e.g. from 300 to5000 daltons, such as DPPE-PEG or DSPE-PEG, i.e. DPPE (or DSPE) having aPEG polymer attached thereto. For example, DPPE-PEG2000 refers to DPPEhaving attached thereto a PEG polymer having a mean average molecularweight of about 2000. As explained in detail in the following, thesePEG-modified phospholipids are preferably used in combination withnon-modified phospholipids.

Both neutral and charged phospholipids can satisfactorily be employed inthe process of the present invention, as well as mixtures thereof. Asused herein and in the prior art, the term “charged” in relation with“phospholipids” means that the individual phospholipid molecules have anoverall net charge, be it positive or, more frequently, negative.

Examples of phospholipids bearing an overall negative charge arederivatives, in particular fatty acid di-esters, of phosphatidylserine,such as DMPS, DPPS, DSPS; of phosphatidic acid, such as DMPA, DPPA,DSPA; of phosphatidylglycerol such as DMPG, DPPG and DSPG. Also modifiedphospholipids, in particular PEG-modified phosphatidylethanolamines,such as DMPE-PEG750, DMPE-PEG1000, DMPE-PEG2000, DMPE-PEG3000,DMPE-PEG4000, DMPE-PEG5000, DPPE-PEG750, DPPE-PEG1000, DPPE-PEG2000,DPPE-PEG3000, DPPE-PEG4000, DPPE-PEG5000, DSPE-PEG750, DSPE-PEG1000,DSPE-PEG2000, DSPE-PEG3000, DSPE-PEG4000, DSPE-PEG5000, DAPE-PEG750,DAPE-PEG1000, DAPE-PEG2000, DAPE-PEG3000, DAPE-PEG4000 or DAPE-PEG5000can be used as negatively charged molecules. Also the lyso-form of theabove cited phospholipids, such as lysophosphatidylserine derivatives(e.g. lyso-DMPS, -DPPS or -DSPS), lysophosphatidic acid derivatives(e.g. lyso-DMPA, -DPPA or -DSPA) and lysophosphatidylglycerolderivatives (e.g. lyso-DMPG, -DPPG or -DSPG), can advantageously be usedas negatively charged compound.

Examples of phospholipids bearing an overall positive charge arederivatives of ethylphosphatidylcholine, in particular esters ofethylphosphatidylcholine with fatty acids, such as1,2-Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC or DSEPC),1,2-Dipalmitoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DPPC or DPEPC).

Preferably, blends of two or more phospholipids, at least one with aneutral charge and at least one with an overall net charge, areemployed. More preferably, blends of two or more phospholipids, at leastone with neutral and at least one with negative charge are employed. Theamount of charged phospholipid, may vary from about 95% to about 5% byweight, with respect to the total amount of phospholipid, preferablyfrom 80% to 20% by weight. The presence of at least minor amounts, suchas 5% to 20% by wt. with respect to the total weight of phospholipid, ofa (negatively) charged phospholipid may help preventing aggregation ofbubbles or emulsion droplets. It is however possible to use a singlephospholipid, neutral or charged, or a blend of two or morephospholipids, all neutral or all with an overall net charge.

Preferred phospholipids are DAPC, DPPA, DSPA, DMPS, DPPS, DSPS, DPPE,DSPE, DSPG, DPPG and Ethyl-DSPC. Most preferred are DSPA, DPPS or DSPS.

Preferred mixtures of phospholipids are mixtures of DPPS with DPPC, DSPCor DAPC (from 95/5 to 5/95 w/w), mixtures of DSPA with DSPC or DAPC(from 95/5 to 5/95 w/w), mixtures or DSPG or DPPG with DSPC or mixturesof DSPC with Ethyl-DSPC. Most preferred are mixtures of DPPS/DSPC (from50/50 to 10/90 w/w) or DSPA/DSPC (from 50/50 to 20/80 w/w).

The amount of phospholipid is generally comprised between about 0.005and about 1.0% by weight with respect to the total weight of theemulsified mixture. Larger amounts might of course be employed butconsidering that the end product is an injectable contrast agent, it ispreferred not to use excess of additives unless strictly necessary toprovide for a stable and suitable product. In general, by using anamount of phospholipid larger than that indicated as the upper limit ofthe above range, essentially no or a very negligible improvement isobserved in terms of bubble population, bubble size distribution, andbubble stability. Typically, higher amounts of phospholipid are requiredwhen higher volumes of organic solvent are used. Thus, when the volumeof organic solvent amounts to about 50% the volume of the water phase,an amount of about 1% w/w of phospholipid can advantageously be added tothe emulsion. Preferably the amount of phospholipid is comprised between0.01 and 1.0% by weight with respect to the total weight of theemulsified mixture and more preferably between about 0.05% and 0.5% byweight.

As mentioned before, the microbubbles produced according to the processof the invention are stabilized predominantly by a phospholipid, asabove defined. In particular, the envelope surrounding the gas filledmicrobubbles is formed by more than 50% (w/w), preferably by at least80%, and much more preferably by at least 90% of a phospholipid materialas above defined. Conveniently, the substantial totality of thestabilizing envelope of the microbubbles is formed by a phospholipid.

Other amphiphilic materials can however be admixed with thephospholipids forming the stabilizing envelope of the gas-filledmicrobubbles, in amounts of less than 50% of the total weight of theemulsifying composition.

Examples of suitable additional envelope-stabilizing amphiphilicmaterials include, for instance, lysolipids; fatty acids, such aspalmitic acid, stearic acid, lauric acid, myristic acid, arachidic acid,arachidonic acid, behenic acid, oleic acid, linoleic acid or linolenicacid, and their respective salts with alkali or alkali metals; lipidsbearing polymers, such as chitin, hyaluronic acid, polyvinylpyrrolidoneor polyethylene glycol (PEG), also referred as “pegylated lipids”;lipids bearing sulfonated mono- di-, oligo- or polysaccharides; lipidswith ether or ester-linked fatty acids; polymerized lipids; diacetylphosphate; dicetyl phosphate; stearylamine; ceramides; polyoxyethylenefatty acid esters (such as polyoxyethylene fatty acid stearates);polyoxyethylene fatty alcohols; polyoxyethylene fatty alcohol ethers;polyoxyethylated sorbitan fatty acid esters; glycerol polyethyleneglycol ricinoleate; ethoxylated soybean sterols; ethoxylated castor oil;ethylene oxide (EO) and propylene oxide (PO) block copolymers; sterolesters of sugar acids including cholesterol glucuronides, lanosterolglucoronides, 7-dehydrocholesterol glucoronide, ergosterol glucoronide,cholesterol gluconate, lanosterol gluconate, or ergosterol gluconate;esters of sugar acids and alcohols including lauryl glucoronide,stearoyl glucoronide, myristoyl glucoronide, lauryl gluconate, myristoylgluconate, or stearoyl gluconate; esters of sugars with aliphatic acidsincluding sucrose laurate, fructose laurate, sucrose palmitate, sucrosestearate, glucuronic acid, gluconic acid or polyuronic acid; esters ofglycerol with (C₁₂-C₂₄), preferably (C₁₄-C₂₂) dicarboxylic fatty acidsand their respective salts with alkali or alkali-metal salts, such as1,2-dipalmitoyl-sn-3-succinylglycerol or1,3-dipalmitoyl-2-succinylglycerol; saponins including sarsasapogenin,smilagenin, hederagenin, oleanolic acid, or digitoxigenin; long chain(C₁₂-C₂₄) alcohols, including n-decyl alcohol, lauryl alcohol, myristylalcohol, cetyl alcohol, or n-octadecyl alcohol;6-(5-cholesten-3,-yloxy)-1-thio-β-D-galactopyranoside;digalactosyldiglyceride;6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxy-1-thio-β-D-galactopyranoside;6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxyl-1-thio-β-D-mannopyranoside;12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoicacid;N-[12-(((7′-diethylaminocoumarin-3-yl)carbonyl)-methylamino)octadecanoyl]-2-aminopalmiticacid; N-succinyldioleylphosphatidylethanolamine;1-hexadecyl-2-palmitoylglycerophosphoethanolamine;palmitoylhomocysteine; alkylammonium salts comprising at least one(C₁₀-C₂₀), preferably (C₁₄-C₁₈), alkyl chain, such as, for instance,stearylammonium chloride, hexadecylammonium chloride,dimethyldioctadecylammonium bromide (DDAB), hexadecyltrimethylammoniumbromide (CTAB); tertiary or quaternary ammonium salts comprising one orpreferably two (C₁₀-C₂₀), preferably (C₁₄-C₁₈), acyl ester residue, suchas, for instance, 1,2-distearoyl-3-trimethylammonium-propane (DSTAP),1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP),1,2-oleoyl-3-trimethylammonium-propane (DOTAP),1,2-distearoyl-3-dimethylammonium-propane (DSDAP): and mixtures orcombinations thereof.

Small amounts of fatty acids and lyso forms of the phospholipids mayalso form as degradation products of the original phospholipid products,e.g. as a consequence of heating the emulsion.

Preferred additional envelope-stabilizing amphiphilic materials arethose compounds comprising one or two fatty acid residues in theirmolecule, in particular one or two linear (C₁₀-C₂₀)-acyl, preferably(C₁₄-C₁₈)-acyl chains, such as, for instance, the above listed fattyacids, their respective salts and derivatives.

Particularly preferred additional envelope-stabilizing amphiphilicmaterials are those compounds capable of conferring an overall netcharge to the stabilizing envelope, i.e. compounds bearing an overallpositive or negative net charge. Examples of suitable negatively ofpositively charged compounds are, for instance, lyso-phospholipids, i.e.the lyso-form of the above cited phospholipids, such aslysophosphatidylserine derivatives (e.g. lyso-DMPS, -DPPS or -DSPS),lysophosphatidic acid derivatives (e.g. lyso-DMPA, -DPPA or -DSPA) andlysophosphatidylglycerol derivatives (e.g. lyso-DMPG, -DPPG or -DSPG);bile acid salts such as cholic acid salts, deoxycholic acid salts orglycocholic acid salts; (C₁₂-C₂₄), preferably (C₁₄-C₂₂) fatty acid saltssuch as, for instance, palmitic acid salt, stearic acid salt,1,2-dipalmitoyl-sn-3-succinylglycerol salt or1,3-dipalmitoyl-2-succinylglycerol salt; alkylammonium salts with ahalogen counter ion (e.g. chlorine or bromine) comprising at least one(C₁₀-C₂₀) alkyl chain, preferably (C₁₄-C₁₈) alkyl chain, such as, forinstance stearylammonium chloride, hexadecylammonium chloride,dimethyldioctadecylammonium bromide (DDAB), hexadecyltrimethylammoniumbromide (CTAB); tertiary or quaternary ammonium salts with a halogencounter ion (e.g. chlorine or bromine) comprising one or preferably two(C₁₀-C₂₀) acyl chain, preferably (C₁₄-C₁₈) acyl ester residue, such as,for instance, 1,2-distearoyl-3-trimethylammonium-propane (DSTAP),1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP),1,2-oleoyl-3-trimethylammonium-propane (DOTAP),1,2-distearoyl-3-dimethylammonium-propane (DSDAP).

If it is desired to obtain “targeted” ultrasound contrast agents, i.e.contrast agents containing microbubbles that could selectively bind to aspecific site after in vitro or in vivo administration, according to theprocess of the present invention it is also possible to start directlyfrom a phospholipid at least part of which has been modified by theintroduction of a suitably selected targeting ligand or alternatively,and preferably, starting from phospholipid at least part of whichcontain a possibly protected reactive group capable of being coupled ata later stage with the suitably selected targeting ligand containing acomplementary reactive function (e.g. avidin-biotin link).

Therefore, in this specific context, the term “phospholipid” is intendedto encompass both modified and unmodified phospholipids, thus includingphospholipids modified by linking a targeting ligand or a protectivereactive group to the amphiphilic molecule of the phospholipid.

The term “targeting ligand” includes within its meaning any compound,moiety or residue having, or being capable to promote, a targetingactivity of the microbubbles of the invention towards any biological orpathological site within a living body. Materials or substances whichmay serve as targeting ligands include, for example, but are not limitedto proteins, including antibodies, antibody fragments, receptormolecules, receptor binding molecules, glycoproteins and lectins;peptides, including oligopeptides and polypeptides; peptidomimetics;saccharides, including mono and polysaccharides; vitamins; steroids,steroid analogs, hormones, cofactors, bioactive agents and geneticmaterial, including nucleosides, nucleotides and polynucleotides.Targets to which targeting ligand may be associated include tissues suchas, for instance, myocardial tissue (including myocardial cells andcardiomyocites), membranous tissues (including endothelium andepithelium), laminae, connective tissue (including interstitial tissue)or tumors; blood clots; and receptors such as, for instance,cell-surface receptors for peptide hormones, neurotransmitters,antigens, complement fragments, and immunoglobulins and cytoplasmicreceptors for steroid hormones.

Examples of suitable targets and targeting ligands are disclosed, forinstance, in U.S. Pat. No. 6,139,819, which is herein incorporated byreference.

In one preferred embodiment the targeting ligands can be bound to theamphiphilic molecules forming the stabilizing envelope through acovalent bond.

In such a case the specific reactive moiety that needs to be present inthe phospholipid or lipid molecule when a targeting amphiphilic moleculeis desired, will depend on the particular targeting ligand to be coupledthereto. As an example, if the targeting ligand can be linked to theamphiphilic molecule through an amino group, suitable reactive moietiesfor the amphiphilic molecule may be isothiocyanate groups (that willform a thiourea bond), reactive esters (to form an amide bond), aldehydegroups (for the formation of an imine bond to be reduced to analkylamine bond), etc.; if the targeting ligand can be linked to theamphiphilic molecule through a thiol group, suitable complementaryreactive moieties for the amphiphilic molecule include haloacetylderivatives or maleimides (to form a thioether bond); and if thetargeting ligand can be linked to the amphiphilic molecule through acarboxylic group, suitable reactive moieties for the amphiphilicmolecule might be amines and hydrazides (to form amide or alkylamidebonds). The reactive moiety can be linked either directly to thephospholipid molecule or to a modifying moiety (e.g. PEG) linked to thephospholipid.

As indicated above, in a preferred embodiment, when a contrast agentcontaining targeted microbubbles is desired, at least part of thestarting phospholipid will contain a suitable reactive moiety and thetargeting ligand containing the complementary functionality will belinked thereto either at any step before the lyophilization, by addingthe targeting ligand containing the complementary functionality into thephase containing the functionalised phospholipids/lipids, either before,during or after the generation of the emulsion, or just before thereconstitution step. In this latter case it would be possible to fullyexploit the flexibility of the system as the microbubbles containing atleast part of the film-forming phospholipids, or of the associatedlipids, suitably functionalised, might then be bound to any desiredtargeting ligand, sharing the same reactive complementary group.

Not necessarily however the targeting ligand needs to be bound to theamphiphilic molecules through a covalent bond. The targeting ligands mayalso be suitably associated to the microbubbles via physical and/orelectrostatic types of interactions. As an example, a functional moietyhaving a high affinity and selectivity for a complementary moiety can beintroduced into the phospholipid molecule, while the complementarymoiety will be linked to the targeting ligand. For instance, an avidin(or streptavidin) moiety (having high affinity for biotin) can becovalently linked to a microbubble stabilizing phospholipid while thecomplementary biotin moiety can be incorporated into a suitabletargeting ligand, e.g. a peptide or an antibody. The biotin-labelledtargeting ligand will thus be associated to the avidin-labelledmicrobubble by means of the avidin-biotin coupling system. According toan alternative embodiment, a biotin-containing phospholipid can be usedas a compound to form the stabilizing envelope of a microbubble;biotin-containing phospholipid incorporated in the stabilizing envelopeis then reacted first with avidin (or neutravidin) and then with abiotin-containing ligand. Examples of biotin/avidin labelling ofphospholipids and peptides are also disclosed in the above cited U.S.Pat. No. 6,139,819. Alternatively, van der Waal's interactions,electrostatic interactions and other association processes may associateor bind the targeting ligand to the amphiphilic molecules.

Examples of suitable specific targets to which the microbubbles of theinvention can be directed are, for instance, fibrin, the α_(v)β₃receptor or the GPIIbIIIa receptor on activated platelets. Fibrin andplatelets are in fact generally present in “thrombi”, i.e. coagula whichmay form in the blood stream and cause a vascular obstruction. Suitablebinding peptides are disclosed, for instance, in the above cited U.S.Pat. No. 6,139,819. Further binding peptides specific forfibrin-targeting are disclosed, for instance, in International patentapplication WO 02/055544, which is herein incorporated by reference.

Other examples of important targets include receptors in vulnerableplaques and tumor specific receptors, such as kinase domain region (KDR)and VEGF (vascular endothelial growth factor)/KDR complex. Bindingpeptides suitable for KDR or VEGF/KDR complex are disclosed, forinstance, in International Patent application WO 03/74005 and WO03/084574, both herein incorporated by reference.

Process

The emulsifying step a) of the process of the present invention can becarried out by submitting the aqueous medium and the core solvent in thepresence of at least one phospholipid to any appropriateemulsion-generating technique known in the art, such as, for instance,sonication, shaking, high pressure homogenization, micromixing, membraneemulsification, high speed stirring or high shear mixing, e.g. using arotor-stator homogenizer. For instance, a rotor-stator homogenizer isemployed, such as Polytron® PT3000. The agitation speed of therotor-stator homogenizer can be selected depending from the componentsof the emulsion, the volume of the emulsion and of the diameter of thevessel containing the emulsion and the desired final diameter of themicrodroplets of solvent in the emulsion. In general, it has beenobserved that, when using a rotor-stator homogenizer having a probe ofabout 3 cm diameter immersed in a 50-80 ml mixture contained in 3.5-5 cmdiameter beaker, an agitation speed of about 8000 rpm is typicallysufficient to obtain microdroplets having a mean numerical diametersufficiently reduced to result, after lyophilization and reconstitutionof the lyophilized matrix, in gas-filled microbubbles having a diameterof less than about 1.8 μm. By increasing the agitation speed at about12000 rpm, it is in general possible to obtain gas-filled microbubbleshaving a number mean diameter of less than about 1.5 μm, while with anagitation speed of about 14000-15000 rpm, gas-filled microbubbles havinga number mean diameter of about 1.0 μm or less can generally beobtained. In general it has been observed that by increasing theagitation speed above about 18000 rpm, slight further reduction ofmicrobubbles size is obtained.

Alternatively, a micromixing technique can also be employed foremulsifying the mixture. As known, a micromixer typically contain atleast two inlets and at least one outlet. The organic solvent is thusintroduced into the mixer through a first inlet (at a flow rate of e.g.0.05-5 ml/min), while the aqueous phase is introduced through the secondinlet (e.g. at a flow rate of 2-100 ml/min). The outlet of themicromixer is then connected to the vessel containing the aqueous, sothat the aqueous phase drawn from said vessel at subsequent instants andintroduced into the micromixer contains increasing amounts of emulsifiedsolvent. When the whole volume of solvent has been added, the emulsionfrom the container can be kept under recirculation through themicromixer for a further predetermined period of time, e.g. 5-120minutes, to allow completion of the emulsion.

Depending on the emulsion technique, the organic solvent can beintroduced gradually during the emulsification step or at once beforestarting the emulsification step. Alternatively the aqueous medium maybe gradually added to the water immiscible solvent during theemulsification step or at once before starting the emulsification step.The phospholipid can be either dispersed in the aqueous medium or in theorganic solvent, before admixing the two, or it may be separately addedthe aqueous-organic mixture before or during the emulsification step.Preferably, the phospholipid is dispersed in the organic solvent(preferably cyclooctane).

The emulsification of step a) is conveniently carried out at roomtemperature, e.g. at a temperature of 22° C.±5° C., or at highertemperatures, for instance 50° C.-60° C. (e.g. in the case of coresolvents with high boiling points) or at lower temperature, for instance0° C.-10° C. (e.g. in the case of core solvents with boiling pointsclose to room temperature). The temperature is preferably kept below theboiling temperature of the organic solvent, preferably at least 5° C.below said temperature, more preferably at least 10° C. below. Asprolonged exposure of the mixture at high temperatures (e.g. 90° C. ormore) may cause possible degradations of phospholipids, with consequentformation of of the respective lyso-derivatives, it is in generalpreferred to avoid such prolonged heating at high temperatures.

If necessary, the aqueous medium containing the phospholipids can besubjected to controlled heating, in order to facilitate the dispersionthereof. For instance, the phospholipid containing aqueous suspensioncan be heated at about 60-70° C. for about 15 minutes and then allowedto cool at the temperature at which the emulsion step is then carriedout.

As previously mentioned, additional amphiphilic materials, such as thosepreviously listed, can also be introduced into the emulsifying mixturecontaining the phospholipid. The amount of said additional amphiphiliccompounds is preferably not higher than about 50% by weight with respectto the total weight of amphiphilic material, more preferably not higherthan 20% by weight, down to an amount of e.g. about 0.1%.

The aqueous medium may, if desired, further contain one or moreexcipients.

As used herein, the term “excipient” refers to any additive useful inthe present invention, such as those additives employed to increase thestability of the emulsion or of the lyophilisate intermediate and/or toprovide for pharmaceutically acceptable and stable final compositions.

Exemplary excipients in this regard are, for instance, viscosityenhancers and/or solubility aids for the phospholipids.

Viscosity enhancers and solubility aids that may suitably be employedare for example mono- or polysaccharides, such as glucose, lactose,saccharose, and dextrans, aliphatic alcohols, such as isopropyl alcoholand butyl alcohol, polyols such as glycerol, 1,2-propanediol, and thelike agents. In general however we have found that it is unnecessary toincorporate additives such as viscosity enhancers, which are commonlyemployed in many existing contrast agent formulations, into the contrastagents of the present invention. This is a further advantage of thepresent invention as the number of components administered to the bodyof a subject is kept to a minimum and the viscosity of the contrastagents is maintained as low as possible.

As mentioned before, the Applicant has found substantially unnecessary,to add water-insoluble structure-builders, such as cholesterol, to theemulsifying mixture. As a matter of fact, it has been observed that anamount of 0.05% (w/w with respect to the total weight of the emulsifyingmixture) of cholesterol dramatically reduces the conversion yield frommicrodroplets into gas-filled microvesicles, further resulting in abroad-dispersion of the vesicles' size. The amount of water-insolublecompounds in the emulsifying mixture, particularly of those compoundsnot comprising one or two fatty acid residue in their structure, is thuspreferably lower than 0.050%, more preferably lower than about 0.030% byweight with respect to the total weight of the emulsion.

Emulsions produced according to step a) may advantageously be subjectedto one or more washing steps, prior to the lyophilization of step b), inorder to remove excess of phospholipids in the aqueous phase (notassociated to the emulsion) and separate and remove optional additivessuch as viscosity enhancers and solubility aids, as well as undesiredmaterial such as colloidal particles, and undersized and/or oversizedemulsion droplets. Such washing may be effected in per se known manner,the emulsion being separated using techniques such as decantation,flotation, centrifugation, cross flow filtration and the like.

If washing steps are foreseen, and if a lyoprotective agent was presentin the original aqueous phase prior to the generation of the emulsion,said washing steps can be performed with aqueous solutions containingone or more lyoprotective agents to replace the amount of lyoprotectiveagents partially removed with the washings. On the other side, if nolyoprotectant was present in the emulsified aqueous-organic mixture, theformed emulsion can be washed with a lyoprotectant-containing aqueoussolution, in order to introduce the lyoprotectant into the emulsifiedmixture or, alternatively, the lyoprotectant can be added after thewashing steps, prior to lyophilisation.

If desired, the emulsion (either as such or after the washing step) canbe subjected to a ultrafiltration or microfiltration step beforelyophilization, in order to further reduce the amount of large sizemicrobubbles in the final reconstituted suspension. Duringmicrofiltration, e.g. with a 5 μm or 3 μm filter, large sizemicrodroplets are in fact retained by the filter and separated from therest of the small size microdroplets, thus preventing the formation oflarge size microbubbles upon reconstitution of the lyophilized material.Microfiltration can be accomplished according to conventional techniquessuch as positive filtration, vacuum filtration or in-line filtration.Membranes of filtration can be Nylon, glass fiber, cellulose, paper,polycarbonate or polyester (Nuclepore®) membranes.

According to an alternative embodiment, an additional amphiphiliccompound can be added after the formation of the emulsion according tothe above teachings, either with or without the washing steps. Inparticular, an aqueous suspension of the desired compound is added tothe formed emulsion, preferably under agitation and heating (preferablyat less than 80° C., e.g. 40° C.-80° C., in particular 50-70° C.), inorder to add said compound to the stabilizing envelope. This alternativeembodiment is particularly useful to subsequently introduce into thestabilizing layer amphiphilic compounds which may otherwise negativelyaffect the properties of the final product if introduced in the initialmixture of the emulsion. Examples of amphiphilic compounds which canconveniently be subsequently introduced as additional components of thestabilizing envelope after the preparation of the initial emulsion are,for instance, PEG-modified phospholipids, in particular PEG-modifiedphosphatidylethanolamines, such as DMPE-PEG750, DMPE-PEG1000,DMPE-PEG2000, DMPE-PEG3000, DMPE-PEG4000, DMPE-PEG5000, DPPE-PEG750,DPPE-PEG1000, DPPE-PEG2000, DPPE-PEG3000, DPPE-PEG4000, DPPE-PEG5000,DSPE-PEG750, DSPE-PEG1000, DSPE-PEG2000, DSPE-PEG3000, DSPE-PEG4000,DSPE-PEG5000, DAPE-PEG750, DAPE-PEG1000, DAPE-PEG2000, DAPE-PEG3000,DAPE-PEG4000 or DAPE-PEG5000. Similarly, also PEG-modified phospholipidsbearing reactive moieties or targeting ligands (e.g. containing biotin,maleimide, or maleimide-peptide) can conveniently be introducedsubsequently according to this method. In addition, this technique canalso be used to subsequently add to the composition of the stabilizinglayer other components, such as lipopeptides or polymeric surfactants.Examples of polymeric surfactants which can be conveniently added afterformation of the emulsion are, for instance, ethyleneoxide-propylenoxideblock copolymers, such as Pluronic F68, Pluronic F108, Pluronic F-127(Sigma Aldrich, Missouri, USA); Polyoxyethylated alkyl ethers such asBrij® 78 (Sigma Aldrich, Missouri, USA); Polyoxyethylene fatty acidesters such as Myrj® 53 or Myrj® 59 (Sigma Aldrich, Missouri, USA);Polyoxyethylenesorbitan fatty acid ester such as Tween® 60 (SigmaAldrich, Missouri, USA); or Polyethylene glycol tert-octylphenyl ethersuch as Triton®X-100 (Sigma Aldrich, Missouri, USA).

The Applicant has in fact observed that the use of a mixture containinglimited amounts (e.g. less than 10% by weight) of a PEG modifiedphospholipid (e.g. DSPE-PEG or DPPE-PEG) together with a film formingphospholipid (e.g. DPPS or a 50:50 mixture of DAPC/DPPS) for preparingan emulsion according to the process of the invention, may determine asubstantial broadening of the size distribution in the final product,with respect to the size distribution of microbubbles obtained from anemulsion containing only the film forming phospholipid. On the otherside, if an emulsion containing only the film forming phospholipid isfirst prepared and then an aqueous suspension of the PEG modifiedphospholipid is subsequently added to the obtained emulsion (e.g. underagitation for 1 hour, at a temperature of about 60° C.), it has beenobserved that a rather high amount (typically more than 30% by weight)of the PEG modified phospholipid can be incorporated into thestabilizing envelope, without substantially affecting the sizedistribution of the final product.

According to a preferred embodiment, the emulsion is subjected to acontrolled additional heating treatment before the lyophilization step.The additional heating of the emulsion is preferably performed into asealed container. The heat treatment can vary from about 15 minutes toabout 90 minutes, at temperatures comprised from about 60° C. to about125° C., preferably from about 80° C. to about 120° C. In general, thehigher the temperature, the shortest the time of the thermal treatment.During the heating, the emulsion can optionally be kept under agitation.

As observed by the Applicant, while this additional thermal treatmentmay result in a partial degradation of the phospholipids (e.g. with acontent of about 5-20% w/w of lysolipids in the final product, when theemulsion is heated at about 100-120° C. for about 30 min), it hasnevertheless the great advantage of allowing a substantial narrowing ofthe size distribution and an increase of the total number ofmicrobubbles in the final suspension, independently from the workingconditions of the initial emulsification step (e.g. type of organicsolvent, emulsifying technique, optional washing steps, etc.).

The thermally treated emulsion can then be directly subjected tolyophilization, typically without the need of further washing steps.

Lyophilization of the emulsion according to step b) may be carried outby initially freezing the emulsion and thereafter lyophilizing thefrozen emulsion, by per se generally known methods and devices. Sincethe dried, lyophilized, product will normally be reconstituted byaddition of a carrier liquid prior to administration, the emulsion mayadvantageously be filled into sealable vials prior to lyophilization soas to give vials each containing an appropriate amount, e.g. a singledosage unit, of lyophilized dried product for reconstitution into aninjectable form. By lyophilizing the emulsion in individual vials ratherthan in bulk, handling of the delicate honeycomb-like structure of thelyophilized product and the risk of at least partially degrading thisstructure are avoided.

Following lyophilization, the vacuum can be removed in the lyophilizerby introducing the desired gas to form the microbubbles in the finalformulation of the contrast agent. This will allow to fill the headspaceof the vials with the desired gas and then seal the vials with anappropriate closure. Alternatively, the vial can be kept under vacuumand sealed, while the gas is added at a later stage, e.g. just beforeadministration, for instance when the gas is a radioactive or ahyperpolarized gas.

The so obtained lyophilized product in the presence of the suitable gascan thus be stably stored for several months before being reconstitutedby dissolving it into an aqueous carrier liquid, to obtain a suspensionof gas-filled microbubbles.

Any biocompatible gas, gas precursor or mixture thereof may be employedto fill the above microvesicles, the gas being selected depending on thechosen modality.

The gas may comprise, for example, air; nitrogen; oxygen; carbondioxide; hydrogen; nitrous oxide; a noble or inert gas such as helium,argon, xenon or krypton; a radioactive gas such as Xe¹³³ or Kr⁸¹; ahyperpolarized noble gas such as hyperpolarized helium, hyperpolarizedxenon or hyperpolarized neon; a low molecular weight hydrocarbon (e.g.containing up to 7 carbon atoms), for example an alkane such as methane,ethane, propane, butane, isobutane, pentane or isopentane, a cycloalkanesuch as cyclobutane or cyclopentane, an alkene such as propene, buteneor isobutene, or an alkyne such as acetylene; an ether; a ketone; anester; halogenated gases, preferably fluorinated gases, such as orhalogenated, fluorinated or perfluorinated low molecular weighthydrocarbons (e.g. containing up to 7 carbon atoms); or a mixture of anyof the foregoing. Where a halogenated hydrocarbon is used, preferably atleast some, more preferably all, of the halogen atoms in said compoundare fluorine atoms.

Fluorinated gases are preferred, in particular perfluorinated gases,especially in the field of ultrasound imaging. Fluorinated gases includematerials which contain at least one fluorine atom such as, for instancefluorinated hydrocarbons (organic compounds containing one or morecarbon atoms and fluorine); sulfur hexafluoride; fluorinated, preferablyperfluorinated, ketones such as perfluoroacetone; and fluorinated,preferably perfluorinated, ethers such as perfluorodiethyl ether.Preferred compounds are perfluorinated gases, such as SF₆ orperfluorocarbons (perfluorinated hydrocarbons), i.e. hydrocarbons whereall the hydrogen atoms are replaced by fluorine atoms, which are knownto form particularly stable microbubble suspensions, as disclosed, forinstance, in EP 0554 213, which is herein incorporated by reference.

The term perfluorocarbon includes saturated, unsaturated, and cyclicperfluorocarbons. Examples of biocompatible, physiologically acceptableperfluorocarbons are: perfluoroalkanes, such as perfluoromethane,perfluoroethane, perfluoropropanes, perfluorobutanes (e.g.perfluoro-n-butane, optionally in admixture with other isomers such asperfluoro-isobutane), perfluoropentanes, perfluorohexanes orperfluoroheptanes; perfluoroalkenes, such as perfluoropropene,perfluorobutenes (e.g. perfluorobut-2ene) or perfluorobutadiene;perfluoroalkynes (e.g. perfluorobut-2-yne); and perfluorocycloalkanes(e.g. perfluorocyclobutane, perfluoromethylcyclobutane,perfluorodimethylcyclobutanes, perfluorotrimethylcyclobutanes,perfluorocyclopentane, perfluoromethylcyclopentane,perfluorodimethylcyclopentanes, perfluorocyclohexane,perfluoromethylcyclohexane and perfluorocycloheptane). Preferredsaturated perfluorocarbons have the formula C_(n)F_(n+2), where n isfrom 1 to 12, preferably from 2 to 10, most preferably from 3 to 8 andeven more preferably from 3 to 6. Suitable perfluorocarbons include, forexample, CF₄, C₂F₆, C₃F₈, C₄F₈, C₄F₁₀, C₅F₁₂, C₆F₁₂, C₆F₁₄, C₇F₁₄, C₇F₆,CF₁₈, and C₉F₂₀.

Particularly preferred gases are SF₆ or perfluorocarbons selected fromCF₄, C₂F₆, C₃F₈, C₄F₈, C₄F₁₀ or mixtures thereof; SF₆, C₃F₈ or C₄F₁₀ areparticularly preferred.

It may also be advantageous to use a mixture of any of the above gasesin any ratio. For instance, the mixture may comprise a conventional gas,such as nitrogen, air or carbon dioxide and a gas forming a stablemicrobubble suspension, such as sulfur hexafluoride or a perfluorocarbonas indicated above. Examples of suitable gas mixtures can be found, forinstance, in WO 94/09829, which is herein incorporated by reference. Thefollowing combinations are particularly preferred: a mixture of gases(A) and (B) in which the gas (B) is a fluorinated gas, preferablyselected from SF₆, CF₄, C₂F₆, C₃F₆, C₃F₈, C₄F₆, C₄F₈, C₄F₁₀, C₅F₁₀,C₅F₁₂ or mixtures thereof, and (A) is selected from air, oxygen,nitrogen, carbon dioxide or mixtures thereof. The amount of gas (B) canrepresent from about 0.5% to about 95% v/v of the total mixture,preferably from about 5% to 80%.

In some instances it may be desirable to include a precursor to agaseous substance (i.e. a material that is capable of being converted toa gas in vivo). Preferably the gaseous precursor and the gas derivedtherefrom are physiologically acceptable. The gaseous precursor may bepH-activated, photo-activated, temperature activated, etc. For example,certain perfluorocarbons may be used as temperature activated gaseousprecursors. These perfluorocarbons, such as perfluoropentane orperfluorohexane, have a liquid/gas phase transition temperature aboveroom temperature (or the temperature at which the agents are producedand/or stored) but below body temperature; thus, they undergo aliquid/gas phase transition and are converted to a gas within the humanbody. Furthermore, he term “gas” as used herein includes mixtures invapor form at the normal human body temperature of 37° C. Compoundswhich at the temperature of 37° C. are liquid may thus also be used inlimited amounts in admixture with other gaseous compounds, to obtain amixture which is in a vapor phase at 37° C.

For ultrasonic echography, the biocompatible gas or gas mixture ispreferably selected from air, nitrogen, carbon dioxide, helium, krypton,xenon, argon, methane, halogenated hydrocarbons (including fluorinatedgases such as perfluorocarbons and sulfur hexafluoride) or mixturesthereof. Advantageously, perfluorocarbons (in particular C₄F₁₀ or C₃F₈)or SF₆ can be used, optionally in admixture with air or nitrogen.

For the use in MRI the microbubbles will preferably contain ahyperpolarized noble gas such as hyperpolarized neon, hyperpolarizedhelium, hyperpolarized xenon, or mixtures thereof, optionally inadmixture with air, CO₂, oxygen, nitrogen, helium, xenon, or any of thehalogenated hydrocarbons as defined above.

For use in scintigraphy, the microbubbles according to the inventionwill preferably contain radioactive gases such as Xe¹³³ or Kr⁸¹ ormixtures thereof, optionally in admixture with air, CO₂, oxygen,nitrogen, helium, kripton or any of the halogenated hydrocarbons asdefined above.

The lyophilized composition in contact with the gas can then be veryeasily reconstituted by the addition of an appropriate sterile aqueousinjectable and physiologically acceptable carrier liquid such as sterilepyrogen-free water for injection, an aqueous solution such as saline(which may advantageously be balanced so that the final product forinjection is not hypotonic), or an aqueous solution of one or moretonicity-adjusting substances such as salts (e.g. of plasma cations withphysiologically tolerable counterions), or sugars, sugar alcohols,glycols and other non-ionic polyol materials (e.g. glucose, sucrose,sorbitol, mannitol, glycerol, polyethylene glycols, propylene glycolsand the like), requiring only minimal agitation such as may, forexample, be provided by gentle hand-shaking.

As observed by the Applicant, the so obtained reconstituted microbubbleshave generally a number mean diameter which is slightly lower than thenumber mean diameter measured for the microdroplets of the emulsion. Themean number diameter of the microbubbles is in general from about 60% toabout 90% of the mean number diameter of the emulsion's microdroplets.In most cases, a mean number diameter of the microbubbles of about70-75% of the mean number diameter of the microdroplets has beenobserved.

Where the dried product is contained in a vial, this is convenientlysealed with a septum through which the carrier liquid may be injectedusing an optionally pre-filled syringe; alternatively the dried productand carrier liquid may be supplied together in a dual chamber devicesuch as a dual chamber syringe. It may be advantageous to mix or gentlyshake the product following reconstitution. However, as noted above, inthe stabilized contrast agents according to the invention the size ofthe gas microbubbles may be substantially independent of the amount ofagitation energy applied to the reconstituted dried product. Accordinglyno more than gentle hand-shaking may be required to give reproducibleproducts with consistent microbubble size.

The microbubble suspensions generated upon reconstitution in water or anaqueous solution may be stable for at least 12 hours, thus permittingconsiderable flexibility as to when the dried product is reconstitutedprior to injection.

Unless it contains a hyperpolarized gas, known to require specialstorage conditions, the lyophilised residue may be stored andtransported without need of temperature control of its environment andin particular it may be supplied to hospitals and physicians for on siteformulation into a ready-to-use administrable suspension withoutrequiring such users to have special storage facilities.

Preferably in such a case it can be supplied in the form of a twocomponent kit.

Said two component kit can include two separate containers or adual-chamber container. In the former case preferably the container is aconventional septum-sealed vial, wherein the vial containing thelyophilized residue of step b) is sealed with a septum through which thecarrier liquid may be injected using an optionally prefilled syringe. Insuch a case the syringe used as the container of the second component isalso used then for injecting the contrast agent. In the latter case,preferably the dual-chamber container is a dual-chamber syringe and oncethe lyophilisate has been reconstituted and then suitably mixed orgently shaken, the container can be used directly for injecting thecontrast agent. In both cases means for directing or permittingapplication of sufficient bubble forming energy into the contents of thecontainer are provided. However, as noted above, in the stabilisedcontrast agents according to the invention the size of the gasmicrobubbles is substantially independent of the amount of agitationenergy applied to the reconstituted dried product. Accordingly no morethan gentle hand shaking is generally required to give reproducibleproducts with consistent microbubble size.

It can be appreciated by one ordinary skilled in the art that othertwo-chamber reconstitution systems capable of combining the dried powderwith the aqueous solution in a sterile manner are also within the scopeof the present invention. In such systems, it is particularlyadvantageous if the aqueous phase can be interposed between thewater-insoluble gas and the environment, to increase shelf life of theproduct. Where a material necessary for forming the contrast agent isnot already present in the container (e.g. a targeting ligand to belinked to the phospholipid during reconstitution), it can be packagedwith the other components of the kit, preferably in a form or containeradapted to facilitate ready combination with the other components of thekit.

No specific containers, vial or connection systems are required; thepresent invention may use conventional containers, vials and adapters.The only requirement is a good seal between the stopper and thecontainer. The quality of the seal, therefore, becomes a matter ofprimary concern; any degradation of seal integrity could allowundesirables substances to enter the vial. In addition to assuringsterility, vacuum retention is essential for products stoppered atambient or reduced pressures to assure safe and proper reconstitution.As to the stopper, it may be a compound or multicomponent formulationbased on an elastomer, such as poly(isobutylene) or butyl rubber.

The contrast agents obtainable by the process of the present inventionmay be used in a variety of diagnostic imaging techniques, including inparticular ultrasound and Magnetic Resonance. Possible other diagnosticimaging applications include scintigraphy, light imaging, and X-rayimaging, including X-ray phase contrast imaging.

Their use in diagnostic ultrasound imaging and in MR imaging, e.g. assusceptibility contrast agents and as hyperpolarized gas bubbles,constitute preferred features of the invention. A variety of imagingtechniques may be employed in ultrasound applications, for exampleincluding fundamental and harmonic B-mode imaging, pulse or phaseinversion imaging and fundamental and harmonic Doppler imaging; ifdesired three-dimensional imaging techniques may be used.

In vivo ultrasound tests in rabbits, dogs and pigs have shown thatcontrast agents according to the invention may produce an increase inbackscattered signal intensity from the myocardium of 15-25 dB followingintravenous injection of doses as low as 0.001 ml/kg body weight.Signals may be observed at even lower doses using more sensitivetechniques such as color Doppler or power pulse inversion. At these lowdoses, attenuation in blood-filled compartments such as the heartchambers has been found to be sufficiently low to permit visualizationof regions of interest in the myocardial vasculature. Tests have alsoshown such intravenously injected contrast agents to be distributedthroughout the whole blood pool, thereby enhancing the echogenicity ofall vascularised tissues, and to be recirculated. They have also beenfound useful as general Doppler signal enhancement aids, and mayadditionally be useful in ultrasound-computed tomography and inphysiologically triggered or intermittent imaging.

For ultrasound applications such as echocardiography, in order to permitfree passage through the pulmonary system and to achieve resonance withthe preferred imaging frequencies of about 0.1-15 MHz, microbubbleshaving an average size of 0.1-10 μm, e.g. 0.5-7 μm are generallyemployed. As described above, contrast agents according to the inventionmay be produced with a very narrow size distribution for the microbubbledispersion within the range preferred for echocardiography, therebygreatly enhancing their echogenicity as well as their safety in vivo,and rendering the contrast agents of particular advantage inapplications such as blood pressure measurements, blood flow tracing andultrasound tomography.

In ultrasound applications the contrast agents of the invention may, forexample, be administered in doses such that the amount of phospholipidinjected is in the range 0.1-200 μg/kg body weight, typically 10-200μg/kg in the absence of a washing step for the emulsion and 0.1-30 μg/kgif the emulsion has been washed prior to lyophilisation. It will beappreciated that the use of such low levels of phospholipid is ofsubstantial advantage in minimising possible toxic side effects.Furthermore, the low levels of phospholipids present in effective dosesmay permit dosage increases to prolong observation times without adverseeffects.

By suitably selecting the components of the mixture and in particularthe amount of agitation energy applied during the emulsion of theaqueous-organic mixture, it is possible to obtain gas-filledmicrobubbles with the desired numerical mean diameter and sizedistribution.

In particular, by exploiting the process according to the presentinvention it is possible to obtain contrast agents comprisingphospholipid-stabilized small-sized gas microbubbles characterized byhaving relatively small mean dimensions and a particularly useful narrowand controlled size distribution.

As known by those skilled in the art, the dimensions of micro/nanoparticles and their respective size distribution can be characterized bya number of parameters, the most frequently used being the mean diameterin number D_(N), the median diameter in number D_(N50), the meandiameter in volume D_(V) and the median diameter in volume D_(V50).While diameters in number provide an indication of the mean numberdimension of the particles, the diameter in volume provides informationon how the total volume of the particles is distributed among the wholepopulation. As the presence of very few large volume particles in apopulation of otherwise small volume particles may cause thecorresponding D_(V) value to be shifted towards high values, it issometimes more convenient to use the D_(V50) value for evaluating thedistribution of a particles' population. D_(V50) is a calculated valueindicating that half of the total of particles' internal volume ispresent in particles having a diameter lower than D_(V50); this allowsto reduce the effects of accidentally formed large volume particles inthe evaluation of the size distribution. Clearly, mono-sized particlesshow identical D_(N), D_(N50), D_(V) and D_(V50) values. On the otherside, an increasing broadening of particles' distribution will result ina larger difference between these various values with a correspondingvariation of the respective ratio thereof (e.g. increase of D_(V)/D_(N)ratio). For example, particles populations containing primarily smallparticles (e.g. particles with a diameter around 2 μm) with neverthelessa small percentage of large particles (for instance particles with adiameter above 8 μm) show higher D_(V) or D_(V50) values as compared tothe D_(N) value, with correspondingly higher D_(V)/D_(N) orD_(V50)/D_(N) ratios. In addition, the microbubbles preparations can becharacterized by the amount of gas contained in microbubbles below apredetermined diameter.

According to an aspect of the present invention, gas-filled microbubblescompositions are provided wherein at least 10% of the total volume ofgas contained in the microbubbles of the composition is contained inmicrovesicles with a diameter of 1.5 μm or less. Preferably, said amountof gas contained in microvesicles with diameter of 1.5 μm or less is atleast 25%, more preferably at least 50% and even more preferably atleast 70%. In some embodiments of the invention, said amount is evenhigehr than 90%. The D_(V50)/D_(N) ratio of the microbubbles compositionis preferably of about 2.10 or lower, more preferably of about 1.80 orlower, much more preferably of about 1.50 or lower. Microbubbles withlower values of the D_(V50)/D_(N) ratio, e.g. 1.20, and even lower, e.g.1.05, can easily be obtained.

Viewed from another aspect, the process of the present invention allowsto prepare microbubbles having a mean diameter in number (D_(N)) of lessthan 1.70 μm and a median diameter in volume (D_(V50)) such that theD_(V50)/D_(N) ratio is of about 2.30 or lower, preferably lower than2.10. Preferably said D_(N) value is of 1.60 μm or lower, morepreferably of 1.50 μm or lower, much more preferably of 1.30 μm orlower. Microbubbles with lower values of D_(N), e.g. of about 1 μm, oreven lower, e.g. 0.85 μm and down to 0.80 μm, can easily be obtainedwith the process of the invention.

The concentration of microbubbles in the reconstituted suspension is ingeneral of at least 1×10⁸ particles per milliliter, preferably of atleast 1×10⁹ particles per milliliter.

The above values of gas amount, D_(V50), D_(N) and number ofmicrobubbles refer to a measurement made by using a Coulter Counter MarkII apparatus fitted with a 30 μm aperture, with a measuring range of 0.7to 20 μm.

This specific category of contrast agents are particularly valuable inultrasound imaging, in particular for imaging techniques relying onnon-linear scattering of microbubbles, as explained below.

Most recent ultrasound contrast-imaging methods exploit the nonlinearscattering characteristics of ultrasound contrast agents. From theliterature (e.g. Eatock et al., Journal of the Acoustical Society ofAmerica, vol. 77(5), pp 1692-1701, 1985) it is known that nonlinearscattering is significant only for microbubbles which are smaller than,or close to, resonance size. In particular, microbubbles with dimensionsof half the resonance size can conveniently be employed. “Half theresonance size” is the size of a microbubble with a resonance frequencythat equals twice the centre frequency of the transmitted ultrasoundwave (which for particular applications may be of up to about 60 MHz).When imaging a volume containing a microbubble-based ultrasound contrastagent, the detectability of the microbubble echoes against tissue echoesis enhanced by the level of nonlinear scattering by the microbubbles,and decreased by the attenuation caused by the microbubbles locatedbetween the probe and the region of interest. Attenuation along thetransmit path reduces the ultrasound-energy available for generatingnonlinear bubble-response; attenuation along the receive path removesecho-energy able to reach the ultrasound probe. In the case of asuspension comprising a wide range of microbubble sizes, themicrobubbles at resonance size, and larger than resonance size, mainlycontribute to transmit-receive attenuation, without contributing in anefficient way to the nonlinear echo signals. Therefore, the overallacoustic response for nonlinear imaging greatly benefits from the use ofa calibrated set of microbubbles having a narrow size distribution and amean size close to half the resonance size. Preferably, microbubblespreparations having a size distribution corresponding to a D_(V50)/D_(N)ratio of about 2.30 or lower, more preferably of 2.10 or lower and muchmore preferably of 2.00 or lower are employed. Preferably, the mean sizeof the employed microbubbles is of about +10% of half the resonancesize, more preferably of about ±5% of half the resonance size.

A yet still further aspect of the present invention thus relates to amethod of diagnostic imaging which comprises administering to a subjecta contrast-enhancing amount of a contrast agent comprising gas-filledmicrobubbles with the size and size distribution as above specified andimaging at least a part of said subject. In particular, said diagnosticimaging includes insonating said subject by means of an ultrasounddevice generating an ultrasound wave with a predetermined transmitfrequency, from which a corresponding resonance size of microbubbles isdetermined, and administering a contrast agent comprising gas-filledmicrobubbles having a narrow size distribution and a mean size close tohalf the resonance size. Preferably, the narrow size distribution and amean size of the microbubbles are as above defined. For instance, a HDI5000 ultrasound machine from Philips (e.g. in pulse inversion mode, withL7-4 probe and Mechanical Index of 0.07), can be used in the diagnosticimaging method. According to this method, said subject is a vertebrateand said contrast agent is introduced into the vasculature or into abody cavity of said vertebrate. Said contrast agent can be supplied as akit, such as those previously described, comprising the lyophilizedproduct in contact with the gas and an aqueous medium forreconstitution.

Embodiments of the Invention Include

1. Methods for preparing a lyophilized matrix which, upon contact withan aqueous carrier liquid and a gas, is reconstitutable into asuspension of gas-filled microbubbles stabilized predominantly by aphospholipid, said method comprising the steps of:

-   -   a) preparing an aqueous-organic emulsion comprising i) an        aqueous medium including water, ii) an organic solvent        substantially immiscible with water; iii) an emulsifying        composition of amphiphilic materials comprising more than 50% by        weight of a phospholipid and iv) a lyoprotecting agent;    -   b) lyophilizing said emulsified mixture, to obtain a lyophilized        matrix comprising said phospholipid.

2. Methods for preparing an injectable contrast agent comprising aliquid aqueous suspension of gas-filled microbubbles stabilizedpredominantly by a phospholipid, which comprises the steps of:

-   -   a) preparing an aqueous-organic emulsion comprising i) an        aqueous medium including water, ii) an organic solvent        substantially immiscible with water; iii) an emulsifying        composition of amphiphilic materials comprising more than 50% by        weight of a phospholipid and iv) a lyoprotecting agent;    -   b) lyophilizing said emulsion, to obtain a lyophilized matrix        comprising said phospholipid;    -   c) contacting said lyophilized matrix with a biocompatible gas;    -   d) reconstituting said lyophilized matrix by dissolving it into        a physiologically acceptable aqueous carrier liquid, to obtain a        suspension of gas-filled microbubbles stabilized predominantly        by said phospholipid.

3. Methods according to embodiments 1 or 2 wherein the step a) ofpreparing the emulsion comprises the following steps:

-   -   a1) preparing a suspension by dispersing the emulsifying        composition and the lyoprotective agent in the aqueous medium;    -   a2) admixing the obtained suspension with the organic solvent;    -   a3) submitting the mixture to controlled agitation, to obtain an        emulsion.

4. Methods according to any of the preceding embodiments, wherein theorganic solvent has a solubility in water of less than 10 g/l.

5. Methods according to any of the preceding embodiments, wherein theorganic solvent has a solubility in water of 1.0 g/l or lower.

6. Methods according to any of the preceding embodiments, wherein theorganic solvent has a solubility in water of 0.2 g/l or lower.

7. Methods according to any of the preceding embodiments, wherein theorganic solvent has a solubility in water of about 0.01 g/l or lower.

8. Methods according to any of the preceding embodiments, wherein theorganic solvent has a solubility in water of 0.001 g/l or lower.

9. Methods according to embodiment 1, wherein the organic solvent isselected among branched or linear alkanes, alkenes, cyclo-alkanes,aromatic hydrocarbons, alkyl ethers, ketones, halogenated hydrocarbons,perfluorinated hydrocarbons and mixtures thereof.

10. Methods according to embodiment 9 wherein the solvent is selectedamong pentane, hexane, heptane, octane, nonane, decane, 1-pentene,2-pentene, 1-octene, cyclopentane, cyclohexane, cyclooctane,1-methyl-cyclohexane, benzene, toluene, ethylbenzene,1,2-dimethylbenzene, 1,3-dimethylbenzene, di-butyl ether anddi-isopropylketone, chloroform, carbon tetrachloride,2-chloro-1-(difluoromethoxy)-1,1,2-trifluoroethane (enflurane),2-chloro-2-(difluoromethoxy)-1,1,1-trifluoroethane (isoflurane),tetrachloro-1,1-difluoroethane, perfluoropentane, perfluorohexane,perfluoroheptane, perfluorononane, perfluorobenzene, perfluorodecalin,methylperfluorobutylether, methylperfluoroisobutylether,ethylperfluorobutylether, ethylperfluoroisobutylether and mixturesthereof

11. Methods according to any of the preceding embodiments, wherein theamount of organic solvent is from about 1% to about 50% by volume withrespect to the amount water.

12. Methods according to any of the preceding embodiments wherein thelyoprotecting agent is selected among carbohydrates, sugar alcohols,polyglycols and mixtures thereof.

13. Methods according to embodiment 12 wherein the lyoprotecting agentis selected among glucose, galactose, fructose, sucrose, trehalose,maltose, lactose, amylose, amylopectin, cyclodextrins, dextran, inuline,soluble starch, hydroxyethyl starch (HES), erythritol, mannitol,sorbitol, polyethyleneglycols and mixtures thereof.

14. Methods according to embodiment 12 or 13 wherein the amount oflyoprotecting agent from about 1% to about 25% by weight with respect tothe weight of water.

15. Methods according to any of embodiments 1, 2 or 3 wherein thephospholipid is selected among dilauroyl-phosphatidylcholine (DLPC),dimyristoyl-phosphatidylcholine (DMPC), dipalmitoyl-phosphatidylcholine(DPPC), diarachidoyl-phosphatidylcholine (DAPC),distearoyl-phosphatidylcholine (DSPC), dioleoyl-phosphatidylcholine(DOPC), 1,2 Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC),dipentadecanoyl-phosphatidylcholine (DPDPC),1-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC),1-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC),1-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC),1-stearoyl-2-palmitoyl-phosphatidylcholine (SPPC),),1-palmitoyl-2-oleylphosphatidylcholine (POPC),1-oleyl-2-palmitoyl-phosphatidylcholine (OPPC),dilauroyl-phosphatidylglycerol (DLPG) and its alkali metal salts,diarachidoylphosphatidyl-glycerol (DAPG) and its alkali metal salts,dimyristoylphosphatidylglycerol (DMPG) and its alkali metal salts,dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal salts,distearoylphosphatidylglycerol (DSPG) and its alkali metal salts,dioleoyl-phosphatidylglycerol (DOPG) and its alkali metal salts,dimyristoyl phosphatidic acid (DMPA) and its alkali metal salts,dipalmitoyl phosphatidic acid (DPPA) and its alkali metal salts,distearoyl phosphatidic acid (DSPA), diarachidoylphosphatidic acid(DAPA) and its alkali metal salts, dimyristoyl-phosphatidylethanolamine(DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidyl-ethanolamine (DSPE), dioleylphosphatidyl-ethanolamine(DOPE), diarachidoylphosphatidylethanolamine (DAPE),dilinoleylphosphatidylethanolamine (DLPE), polyethyleneglycol modifieddimyristoyl-phosphatidylethanolamine (DMPE-PEG), polyethyleneglycolmodified dipalmitoylphosphatidylethanolamine (DPPE-PEG),polyethyleneglycol modified distearoyl phosphatidyl-ethanolamine(DSPE-PEG), polyethyleneglycol modified dioleylphosphatidylethanolamine(DOPE-PEG), polyethyleneglycol modifieddiarachidoylphosphatidylethanolamine (DAPE-PEG), polyethyleneglycolmodified dilinoleylphosphatidylethanolamine (DLPE-PEG), dimyristoylphosphatidylserine (DMPS), diarachidoyl phosphatidylserine (DAPS),dipalmitoyl phosphatidylserine (DPPS), distearoylphosphatidylserine(DSPS), dioleoylphosphatidylserine (DOPS), dipalmitoyl sphingomyelin(DPSP), and distearoylsphingomyelin (DSSP) and mixtures thereof.

16. Methods according to embodiment 1 wherein the emulsifyingcomposition of amphiphilic materials comprises a phospholipid or anamphiphilic material bearing an overall net charge.

17. Methods according to embodiments 1, 2, 3 or 13, wherein the amountof phospholipid is from about 0.005% to about 1.0% by weight withrespect to the total weight of the emulsified mixture.

18. Methods according to embodiment 17 wherein the amount ofphospholipid is from 0.01% to 1.0% by weight with respect to the totalweight of the emulsified mixture.

19. Methods according to embodiment 1, 2 or 3 wherein the phospholipidincludes a targeting ligand or a protective reactive group capable ofreacting with a targeting ligand.

20. Methods according to any of embodiments 1, 2, 3, 15 or 16 whereinthe emulsion further contains an amphiphilic material selected fromlysolipids; fatty acids and their respective salts with alkali or alkalimetals; lipids bearing polymers; lipids bearing sulfonated mono- di-,oligo- or polysaccharides; lipids with ether or ester-linked fattyacids; polymerized lipids; diacetyl phosphate; dicetyl phosphate;stearylamine; ceramides; polyoxyethylene fatty acid esters;polyoxyethylene fatty alcohols; polyoxyethylene fatty alcohol ethers;polyoxyethylated sorbitan fatty acid esters; glycerol polyethyleneglycol ricinoleate; ethoxylated soybean sterols; ethoxylated castor oil;ethylene oxide (EO) and propylene oxide (PO) block copolymers; sterolesters of sugar acids; esters of sugars with aliphatic acids; esters ofglycerol with (C₁₂-C₂₄) dicarboxylic fatty acids and their respectivesalts with alkali or alkali-metal salts; saponins; long chain (C₁₂-C₂₄)alcohols; 6-(5-cholesten-3β-yloxy)-1-thio-β-D-galactopyranoside;digalactosyldiglyceride;6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxy-1-thio-β-D-galactopyranoside;6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxyl-1-thio-β-D-mannopyranoside;12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoicacid;N-[12-(((7′-diethylaminocoumarin-3-yl)carbonyl)-methylamino)octadecanoyl]-2-aminopalmiticacid; N-succinyldioleylphosphatidylethanolamine;1-hexadecyl-2-palmitoylglycerophosphoethanolamine;palmitoylhomocysteine; alkylammonium salts comprising at least one(C₁₀-C₂₀) alkyl chain; tertiary or quaternary ammonium salts comprisingat least one (C₁₀-C₂₀) acyl chain linked to the N-atom through a (C₃-C₆)alkylene bridge: and mixtures or combinations thereof.

21. Methods according to embodiment 1 or 2 wherein the aqueous-organicemulsion of step a) is subjected to a washing step before thelyophilizing step b).

22. Methods according to embodiment 1 or 2 wherein the aqueous-organicemulsion of step a) is subjected to a microfiltration step before thelyophilizing step b).

23. Methods according to embodiment 1 or 2 which further comprisesadding an aqueous suspension comprising a further amphiphilic compoundto the aqueous-organic emulsion obtained according to step a), beforethe lyophilization step b), thus obtaining a second aqueous-organicemulsion comprising said further amphiphilic compound.

24. Methods according to embodiment 23 which further comprises heatingthe mixture of said aqueous suspension and of said aqueous-organicemulsion.

25. Methods according to embodiment 23 wherein said mixture is heated ata temperature of from about 40° C. to about 80° C.

26. Methods according to embodiment 23 wherein said amphiphilic compoundis a PEG-modified phospholipid, a PEG-modified phospholipid bearing areactive moiety or a PEG-modified phospholipid bearing a targetingligand

27. Methods according to embodiments 1, 2 or 23 which further comprises,before the lyophilization step b), subjecting the aqueous-organicemulsion to a controlled heating.

28. Methods according to embodiment 27, wherein said controlled heatingis effected at a temperature of from about 60° C. to 125° C.

29. Methods according to embodiment 28, wherein said controlled heatingis effected at a temperature of from about 80° C. to 120° C.

30. Methods according to embodiment 28, wherein said emulsion iscontained in a sealed vial.

31. Methods according to embodiment 2 or 3 wherein the biocompatible gasis selected among air; nitrogen; oxygen; carbon dioxide; hydrogen;nitrous oxide; inert gases; a low molecular weight hydrocarbon,including a (C₁-C₇) alkane, a (C₄-C₇) cycloalkane, a (C₂-C₇) alkene anda (C₂-C₇) alkyne; an ether; a ketone; an ester; a halogenated (C₁-C₇)hydrocarbon, ketone or ether; or a mixture of any of the foregoing.

32. Methods according to embodiment 31 wherein the halogenatedhydrocarbon gas is selected among bromochlorodifluoro-methane,chlorodifluoromethane, dichlorodifluoro-methane, bromotrifluoromethane,chlorotrifluoromethane, chloropentafluoroethane,dichlorotetrafluoroethane and mixtures thereof.

33. Methods according to embodiment 31 wherein the halogenatedhydrocarbon gas is a perfluorinated hydrocarbon.

34. Methods according to embodiment 33 wherein the perfluorinatedhydrocarbon gas is perfluoromethane, perfluoroethane, aperfluoropropane, a perfluorobutane, a perfluoropentane, aperfluorohexane, a perfluoroheptane; perfluoropropene, aperfluorobutene, perfluorobutadiene, perfluorobut-2-yne,perfluorocyclobutane, perfluoromethylcyclobutane, aperfluorodimethylcyclobutane, a perfluorotrimethylcyclo-butane,perfluorocyclopentane, perfluoromethylcyclopentane, aperfluorodimethylcyclo-pentane, perfluorocyclohexane,perfluoromethylcyclohexane, perfluoromethylcyclohexane and mixturesthereof.

35. Injectable aqueous suspensions of microbubbles filled with abiocompatible gas and comprising a stabilizing layer predominantlycomprising a phospholipid, wherein said microbubbles have a number meandiameter (D_(N)) of less than 1.70 μm and a volume median diameter(D_(V50)) such that the D_(V50)/D_(N) ratio is of about 2.00 or lower.

36. Aqueous suspensions according to embodiment 35 wherein saidmicrobubbles have a D_(N) value of 1.60 μm or lower, preferably of 1.50μm or lower, more preferably of 1.30 μm or lower.

37. Aqueous suspensions according to embodiment 35 wherein saidmicrobubbles have a D_(V50)/D_(N) ratio of about 1.80 or lower,preferably of about 1.60 or lower, more preferably of about 1.50 orlower.

38. Contrast agents for use in diagnostic imaging comprising an aqueoussuspension according to any of embodiments 35 to 37.

39. Methods for diagnostic imaging comprising administering to a subjecta contrast-enhancing amount of an aqueous suspension according to any ofembodiments 35 to 37 and imaging at least a part of said subject.

40. Methods according to embodiment 39 which include insonating saidsubject by means of an ultrasound device generating an ultrasound wavewith a predetermined transmit frequency, from which a correspondingresonance size of microbubbles is determined, and administering acontrast agent comprising gas-filled microbubbles having a narrow sizedistribution and a mean size close to half the resonance size.

The following non-limitative examples are given for better illustratingthe invention.

EXAMPLES

The following materials have been employed in the following examples.

Phospholipids: DPPS Dipalmitoylphosphatidylserine (Genzyme) IUPAC:1,2-Dipalmitoyl-sn- glycero-3-phosphocholine DPPGDipalmitoylphosphatidylglycerol sodium salt (Genzyme) IUPAC: 1,2-Dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] DSPA Distearoylphosphatidic acid sodium salt (Genzyme) IUPAC:1,2-Distearoyl-sn-glycero-3-phosphate DSPGDistearoylphosphatidylglycerol sodium salt (Genzyme) IUPAC:1,2-Distearoyl-sn-glycero-3-phosphoserine) DSPCDistearoylphosphatidylcholine (Genzyme) IUPAC: 1,2-Distearoyl-sn-glycero-3-phosphocholine DSEPC Distearoylethylphosphatidylcholine(Avanti Polar Lipids) IUPAC:1,2-Distearoyl-sn-glycero-3-Ethylphosphocholine DAPCDiarachidoylphosphatidylcholine (Avanti polar Lipids) IUPAC:1,2-Diarachidoyl-sn-glycero-3-phosphocholine DSTAP1,2-Distearoyl-3-trimethylammonium-propane chloride (Avanti PolarLipids) DSPE-PEG2000 Distearoylphosphatidylethanolamine modified withPEG2000, sodium salt (Nektar Therapeutics) DSPE-PEG5000Distearoylphosphatidylethanolamine modified with PEG5000, sodium salt(Nektar Therapeutics) DSPE-PEG2000- Distearoylphosphatidylethanolaminemodified with PEG2000-maleimide maleimid (Avanti Polar lipids) SATAN-Succinimidyl-S-acetylthioacetate (Pierce) RGD-4CH-Ala-Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys-Gly-NH₂ (AnaSpec Inc.)Solvents:

-   Perfluoro-n-hexane (C₆F₁₄), by Fluka-   perfluoromethylcyclohexane (CF₃-cyclo-C₆F₁₁), by Fluka-   perfluoro-n-heptane (C₇F₁₆), by Fluka-   perfluoro-n-nonane (C₉F₂₀), by Aldrich-   perfluorodecalin, by Aldrich-   Cyclohexane, by Fluka-   Cyclooctane, by Fluka-   n-Decane, by Fluka-   n-Octane, by Fluka-   meta xylene, by Fluka-   Diisopropyl cetone, by Fluka-   CCl₄, by Fluka    Lyoprotectants:-   Mannose, by Fluka-   Glucose, by Fluka-   Sorbitol, by Fluka-   Mannitol, by Fluka-   Maltose, by Fluka-   Dextran 6000, by Fluka-   Dextran 15000, by Fluka-   Dextran 40000, by Fluka-   Inulin, by Fluka    Characterization of Microdroplets and Microbubbles.

The size distribution of the emulsions microdroplets has beendetermined:

-   -   a) by means of a Coulter counter (Counter Mark II apparatus        fitted with a 30 μm aperture with a measuring range of 0.7 to 20        μm), when the emulsion has been submitted to a washing step; 10        μl of emulsion were diluted in 100 ml of saline at room        temperature and allowed to equilibrate for 3 minutes prior to        measurement;    -   b) by means of a laser light scattering particle sizer (Malvern        Mastersizer, dilution 200×, focal length 45 mm, standard        presentation), if the emulsion has not been subjected to a        washing step.

The size distributions, volume concentrations and number of themicrobubbles (after lyophilisation and reconstitution with an aqueousphase) were determined by using a Coulter Counter Mark II apparatusfitted with a 30 μm aperture with a measuring range of 0.7 to 20 μm. 50μl of microbubble samples were diluted in 100 ml of saline at roomtemperature and allowed to equilibrate for 3 minutes prior tomeasurement.

The amounts of phospholipids in the final preparatiions (emulsion ofmicrobubbles suspension) were determined by HPLC-MS analysis, with thefollowing set up: Agilent 1100 LC chromatograph, MN CC 125/2 mm-5 C8column from Maherey Nagel, Agilent MSD G1946D detector.

Lyophilization

The lyophilization methodology and apparatus, where not otherwisespecified, were as follows. The emulsion (optionally after the washingstep, if present) is first frozen at −45° C. for 5 minutes and thenfreeze-dried (lyophilized) at room temperature at a pressure of 0.2mbar, by using a Christ-Alpha 2-4 freeze-drier.

Example 1 Preparations 1a-1n

10 mg of DPPS are added to about 10 ml of an 10% (w/w) mannitol aqueoussolution; the suspension is heated at 65° C. for 15 minutes and thencooled at room temperature (22° C.). Perfluoroheptane (8% v/v) is addedto this aqueous phase and emulsified in a beaker of about 4 cm diameterby using a high speed homogenizer (Polytron T3000, probe diameter of 3cm) for 1 minute at the speed indicated in table 1. The resulting mediandiameter in volume (D_(V50)) and a mean diameter in number (D_(N)) ofmicrodroplets of the emulsion are shown in table 1. The emulsion is thencentrifuged (800-1200 rpm for 10 minutes, Sigma centrifuge 3K10) toeliminate the excess of the phospholipid and the separated pellets(microdroplets) were recovered and re-suspended in the same initialvolume of a 10% mannitol aqueous solution.

The washed emulsion is then collected into a 100 ml balloon forlyophilization, frozen and then freeze-dried according to the abovestandard procedure. The lyophilized is then exposed to an atmospherecontaining 35% of perfluoro-n-butane and 65% of nitrogen and thendispersed in a volume of water twice than the initial one by gentle handshaking. The microbubble suspension obtained after reconstitution withdistilled water is analyzed using a Coulter counter. The concentrationof microbubbles in the obtained suspensions was of about 1×10⁹ particlesper ml. The respective microbubbles median diameter in volume (D_(V50)),mean diameter in volume (D_(V)), mean diameter in number (D_(N)) an theamount of microbubbles with diameter larger than 3 μm (percentage overthe total number of microbubbles) are given in table 1. When more thanone example has been performed at the same agitation speed, the valuesindicated in table 1 are referred to the mean calculated value of eachparameter. TABLE 1 EMULSION Gas-filled microbubbles Agitation D_(V50)D_(N) D_(V50) >3 μm <1.5 μm Ex. (rpm) (μm) (μm) (μm) D_(V) (μm) D_(N)(μm) D_(V50)/D_(N) part. % vol. % 1a 8000 4.58 1.77 2.92 3.33 1.51 1.935.44 10.9 1b 9000 4.66 1.94 3.19 3.45 1.53 2.08 6.61 10.0 1c 10000 3.041.74 2.16 2.53 1.33 1.62 1.88 22.6 1d 11000 3.05 1.80 2.17 3.33 1.321.65 1.55 23.7 1e 12000 2.84 1.69 1.86 2.17 1.24 1.50 0.93 33.1 1f 125002.79 1.68 1.75 2.05 1.22 1.44 0.65 35.7 1g 14000 2.20 1.52 1.39 2.451.08 1.29 0.23 58.0 1h 14500 2.00 1.38 1.19 1.39 1.01 1.19 0.06 73.3 1i15000 1.88 1.39 1.22 2.20 1.01 1.21 0.06 70.3 1j 15500 2.19 1.48 1.241.46 1.02 1.22 0.11 68.7 1k 16000 1.83 1.32 1.27 3.08 0.99 1.28 0.1065.2 1l 17000 1.40 1.12 0.91 1.03 0.87 1.05 0.01 95.7

Example 2 Preparations 2a-2j

The same procedure adopted for example 1 is followed, with the onlydifference that the phospholipid is a mixture of DPPS (20% w/w) and DSPC(80% w/w), the total amount of phospholipid remaining unchanged. Theresults are summarized in table 2. TABLE 2 EMULSION Gas-filledmicrobubbles Agitation D_(V50) D_(N) D_(V50) >3 μm <1.5 μm Ex. (rpm)(μm) (μm) (μm) D_(V) (μm) D_(N) (μm) D_(V50)/D_(N) part. % vol. % 2a6000 8.75 3.07 7.55 9.05 2.27 3.33 21.81 1.2 2b 10000 3.54 1.90 3.003.71 1.47 2.04 5.05 11.7 2c 12000 3.04 1.83 2.45 3.73 1.32 1.85 2.1519.8 2d 12500 2.85 1.76 2.21 3.24 1.27 1.74 1.57 24.4 2e 13000 2.98 1.832.25 3.04 1.28 1.76 1.76 23.5 2f 13500 2.91 2.05 1.88 2.46 1.20 1.570.87 33.8 2g 14000 2.45 1.67 1.82 2.66 1.16 1.57 0.57 36.5 2h 14500 2.181.55 1.58 3.04 1.09 1.44 0.38 46.5 2i 15000 1.94 1.42 1.34 1.96 1.041.28 0.31 61.5 2j 16000 1.81 1.38 1.35 2.30 1.03 1.31 0.14 59.0

Example 3 Preparation 3a-3p

The same procedure adopted for examples 2 is followed, with the onlydifference that the DPPS/DSPC weight ratio is varied, as reported intable 3. The results are summarized in table 3. TABLE 3 EMULSIONGas-filled microbubbles DPPS/DSPC Agitation D_(V50) D_(N) D_(V50)D_(N) >3 μm <1.5 μm Ex. ratio (rpm) (μm) (μm) (μm) (μm) D_(V50)/D_(N)part. % vol. % 3a 80/20 12000 2.44 1.54 1.68 1.19 1.41 0.48 39.4 3b75/25 12000 2.53 1.66 1.73 1.18 1.47 0.62 38.3 3c 60/40 11000 3.53 1.862.75 1.45 1.90 4.00 13.6 3d 60/40 12000 2.62 1.60 1.78 1.21 1.47 0.7235.4 3e 60/40 14000 2.36 1.60 1.59 1.13 1.41 0.36 44.7 3f 50/50 120002.81 1.68 2.28 1.30 1.75 2.05 22.6 3g 40/60 11000 3.00 1.72 2.44 1.321.85 2.31 19.2 3h 40/60 12000 2.88 1.75 2.07 1.27 1.63 1.45 25.8 3i40/60 13000 2.61 1.69 1.76 1.16 1.52 0.57 37.6 3j 40/60 14000 2.06 1.431.41 1.07 1.31 0.23 43.8 3k 40/60 14500 2.39 1.67 1.64 1.15 1.43 0.4946.5 3l 30/70 11000 3.12 1.75 2.64 1.37 1.93 2.76 16.3 3m 30/70 120003.08 1.81 2.38 1.34 1.78 2.45 19.7 3n 25/75 11000 3.15 1.85 2.46 1.311.88 2.15 20.7 3o 10/90 11000 3.72 2.26 3.14 1.47 2.13 4.60 12.1 3p 5/95 11000 4.53 2.23 4.08 1.54 2.65 6.35 7.4

Example 4

The same procedure adopted for example 2 is followed, with the onlydifference that mixtures of DSPA and DPPS with different weight ratioswere prepared. The results are summarized in table 4. TABLE 4 EmulsionGas-filled microbubbles Agitation D_(V50) D_(N) D_(V50) D_(N)D_(V50)/ >3 μm <1.5 μm Ex. DSPA/DPPS Ratio (rpm) (μm) (μm) (μm) (μm)D_(N) part. % Vol. % 4a 25/75 12000 2.61 1.63 1.94 1.24 1.56 1.07 30.44b 50/50 11000 2.81 1.86 2.35 1.39 1.69 2.67 18.4 4c 50/50 12000 2.351.57 1.84 1.19 1.55 0.74 34.4 4d 75/25 12000 2.50 1.65 2.11 1.27 1.661.45 25.6

Example 5 Preparations 5a-5i

The same procedure adopted for example 1 is followed, with the onlydifference that a 1/1 (w/w) phospholipid mixture of DPPG and DSPC hasbeen employed (total concentration 1.0 mg/ml) in admixture with 10% w/w(with respect to the total weight of phospholipid) of palmitic acid. Theresults are summarized in table 5. TABLE 5 EMULSION Gas-filledmicrobubbles D_(V50) D_(N) D_(V50) D_(N) >3 μm Ex Agitation (rpm) (μm)(μm) (μm) (μm) D_(V50)/D_(N) part. % <1.5 μm Vol. % 5a 6000 10.02 2.646.87 2.07 3.32 18.00 1.8 5b 8000 5.31 2.49 3.73 1.62 2.30 7.97 7.5 5c9000 5.04 2.69 3.20 1.55 2.06 6.22 9.5 5d 10000 3.82 2.02 2.85 1.38 2.072.65 16.4 5e 10500 3.36 1.96 2.51 1.32 1.89 2.44 20.0 5f 11000 3.22 1.872.31 1.28 1.81 1.41 23.3 5g 12000 2.69 1.61 1.74 1.14 1.53 0.52 39.2 5h13000 2.28 1.56 1.56 1.07 1.46 0.23 47.3 5i 14000 2.00 1.44 1.30 1.001.30 0.26 32.7

Example 6

The same procedure adopted for example 1 is followed, with the onlydifference that DSEPC is used as phospholipid and perfluorohexane isused as the organic solvent. The applied rotation speed is of 11000 rpm.Dimensions, size distribution and percentage of microbubbles withdiameter larger than 3 μm were as follows. D_(V50) (μm) D_(N) (μm)D_(V50)/D_(N) >3 μm (%) 1.65 1.11 1.49 0.30

Example 7 Preparations 7a-7l

Distilled water (10 ml) containing DPPS (10 mg) as phospholipid isheated at 70° C. for 15 minutes and then cooled at room temperature. 0.8ml of an organic solvent as specified in the following table 6 wereemulsified in this aqueous phase using a high speed homogenizer(Polytron T3000) at 10000 rpm for 1 minute. The emulsion is added to 10ml of a 15% dextran 15000 solution, frozen and lyophilized (0.2 mbar, 24hours). After lyophilisation, air is introduced in the lyophilizer. Themicrobubble suspension obtained after reconstitution with distilledwater is analyzed using a Coulter counter. Table 6 summarizes theresults in terms of dimensions and size distribution of microbubbles.TABLE 6 Ex. Solvent D_(V50) (μm) D_(N) (μm) D_(V50)/D_(N) 7a C₆F₁₄ 2.771.44 1.92 7b CF₃-cyclo-C₆F₁₁ 2.24 1.30 1.72 7c C₇F₁₆ 2.48 1.40 1.77 7dC₉F₂₀ 2.46 1.36 1.81 7e perfluorodecalin 3.76 1.52 2.47 7f Cyclohexane2.61 1.41 1.85 7g Cyclooctane 2.43 1.35 1.80 7h Decane 2.01 1.12 1.79 7iOctane 2.87 0.96 2.99 7j meta xylène 2.45 1.21 2.02 7k Diisopropylcetone 1.83 1.05 1.74 7l CCl₄ 1.90 1.27 1.50

Example 8

The above example 7 is repeated with the same methodology, by usingperfluoro hexane as the organic solvent and different lyoprotectingagents at different concentrations as outlined in table 7. Table 7summarizes the results in terms of dimensions and size distribution ofmicrobubbles. TABLE 7 Lyoprotectant and Ex. concentration (w/w) D_(V50)(μm) D_(N) (μm) D_(V50)/D_(N) 8a Mannose 5% 4.35 1.90 2.29 8b Glucose 5%2.59 0.96 2.70 8c Sorbitol 5% 3.84 1.40 2.74 8d Mannitol 10% 2.22 1.221.82 8e Mannitol 5% 2.24 1.21 1.85 8f Mannitol 4% 2.54 1.45 1.75 8gMaltose 5% 3.42 0.99 3.45 8h Dextran 6000 7.5% 3.30 1.48 2.23 8j Dextran15000 5% 2.55 1.31 1.95 8k Dextran 15000 7.5% 2.77 1.44 1.92 8i Dextran40000 7.5% 2.54 1.32 2.29 8l Inulin 5% 3.58 1.43 2.70

Example 9 Preparations 9a-9e

Example 1 is repeated by emulsifying the mixture at a speed of 10000rpm. In addition, the same example is repeated by adding differentamounts of Pluronic F68 (a poloxamer corresponding to Poloxamer 188)into the aqueous phase prior to emulsification, as outlined in table 8.Table 8 shows the results of the comparative experiment, in terms ofsize distribution and conversion yield of the microbubbles. Conversionyield is given as the percentage number of gas-filled microbubblesformed upon reconstitution of the lyophilized matrix with respect to thenumber of microdroplets measured in the emulsion. TABLE 8 Pluronic*Example (mg/ml) D_(V50) D_(N) D_(V50)/D_(N) Conversion yield (%) 9a 02.42 1.38 1.75 28.0 9b 0.25 4.64 1.97 2.36 18.8 9c 0.5 13.85 1.38 10.047.3 9d 1.0 12.59 1.49 8.45 3.2 9e 2.0 15.80 1.23 12.85 0.5*Concentration referred to the volume of aqueous phase

The above results show that with a concentration of poloxamercorresponding to half the concentration of the phospholipid (i.e. about33% of the total amount of surfactants in the mixture), both conversionyields and size distribution of microbubbles are negatively affected.

Example 10 Preparations 10a-10d

Example 9 is repeated, but instead of adding Pluronic F68 to the aqueousphase, different amounts of cholesterol (from Fluka) were added to theorganic phase, prior to emulsification, as outlined in table 9. Table 9shows the results of the comparative experiment, in terms of sizedistribution and conversion yield (from the microdroplets of theemulsion) of the microbubbles. TABLE 9 Cholesterol* Conversion Example(mg/ml) D_(V50) D_(N) D_(V50)/D_(N) yield (%) 10a 0 2.42 1.38 1.75 28.010b 0.10 3.79 1.31 2.89 17.8 10c 0.25 1.35 1.05 1.28 5.7 10d 0.50 14.021.70 8.25 0.8*Concentration referred to the volume of the aqueous phase

The above results show that with a concentration of 0.050% (w/w) ofcholesterol in the aqueous phase, both conversion yield and sizedistribution of microbubbles are highly negatively affected. Aconcentration of 0.025%, while it may provide acceptable dimensions andsize distribution of microbubbles, still results in a rather lowconversion yield.

Example 11

Distilled water (30 ml) containing 60 mg of DPPS and 3 g of mannitol isheated to 70° C. during 15 minutes then cooled to room temperature.

Perfluoroheptane is emulsified in this aqueous phase using a high speedhomogenizer (Polytron®, 12500 rpm, 1 minute).

The resulting emulsion, showing a median diameter in volume (D_(V50)) of2.3 μm and a mean diameter in number (D_(N)) of 2.0 μm, is washed onceby centrifugation, resuspended in 30 ml of a 10% solution of mannitol indistilled water and then divided in three portions (3×10 ml).

The first portion (A) is used as such for the subsequent lyophilizationstep. The second portion (B) is collected into a syringe andhand-injected through a 5 μm Nuclepore® filter (47 mm-Polycarbonate).The third portion (C) is filtered through a 3 μm Nuclepore® filter (47mm-Polycarbonate) with the same method.

The emulsions were frozen in 100 ml balloon (−45° C. for 5 minutes) thenfreeze dried (0.2 mBar, for 72 hours).

Atmospheric pressure is restored by introducing a 35/65 mixture of C₄F₁₀and air. The respective lyophilisates were dispersed in distilled water(10 ml). The so obtained microbubbles suspensions are analysed using aCoulter counter and the results are reported in the following tableD_(V50) D_(N) D_(V50)/D_(N) <1.5 μm Part A 1.71 1.12 1.53 40.2 Part B1.65 1.12 1.47 42.3 Part C 1.57 1.09 1.44 46.3

As shown by the above results, the additional filtration step allows tofurther reduce the dimension of the microbubbles and to reduce therespective size distribution.

Example 12

Example 1 has been repeated, by using 10 mg of a 7/3 (w/w) mixture ofDSPC/DSTAP, at an agitation speed of 11000 rpm. Characterization ofemulsion droplets and microbubbles were as follows: Emulsion dropletsGas-filled microbubbles D_(V50) D_(N) D_(V50) D_(N) >3 μm <1.5 μm 2.361.48 2.10 1.12 0.63 32.7

Example 13

The preparation of example 1 is repeated, by emulsifying the mixture ata speed of 10000 rpm (example 13a).

The same preparation is repeated, by further adding about 0.9 mg ofDSPE-PEG2000 (about 8.3% of the total amount of dispersed phospholipids)to the initial aqueous suspension (example 13b).

No washing by centrifugation is performed on either the twopreparations. Table 10 shows the characterization of the twopreparations, both of the emulsion and of the microbubbles suspension.TABLE 10 Emulsion Microbubbles D_(V50) D_(N) D_(V50) Conversion Example(μm) (μm) (μm) D_(N) (μm) D_(V50)/D_(N) Yield (%) 13a 3.19 1.66 2.661.33 2.00 29.5 13b 4.32 1.43 5.81 1.18 4.92 18.8

The above results show that with a concentration of DSPE-PEG of lessthan 10% by weight (with respect to the total amount of phospholipids),both conversion yields and size distribution of microbubbles arenegatively affected.

Example 14

The preparation of example 11 is repeated, by replacing DPPS with thesame amount of a 1:1 (w/w) mixture of DAPC/DPPS.

The resulting emulsion is divided in three portions of 10 ml, withoutwashing it by centrifugation.

Aqueous suspensions of DSPE-PEG2000 and of DSPE-PEG5000 are separatelyprepared by dispersing 25 mg of the respective DSPE-PEG in 5 ml of a 10%mannitol solution under sonication (3 mm sonication probe, Branson 250sonifier, output 30%, for 5 min).

An aliquot of 2.5 ml of a 10% mannitol solution is then added to a firstportion of the emulsion (example 14a)

An aliquot of 2.5 ml of the prepared DSPE-PEG2000 suspension is added toa second portion of the emulsion (example 14b)

An aliquot of 2.5 ml of the prepared DSPE-PEG5000 suspension is added toa third portion of the emulsion (example 14c)

The three mixtures are heated at 60° C. under stirring for one hour.After cooling at room temperature, the size of microdroplets aredetermined by means of Malvern Mastersizer. Results are reported intable 11.

The emulsions are then freeze dried according to the procedure ofexample 11. Atmospheric pressure is restored by introducing a 35/65mixture of C₄F₁₀ and air. The respective lyophilisates were dispersed indistilled water (10 ml). The so obtained microbubbles suspensions wereanalysed using a Coulter counter (see table 11).

Microbubble suspensions are then washed twice with distilled water bycentrifugation (180 g/10 min) and lyophilized again according to theabove procedure. The amount of DSPE-PEG in the dried composition isdetermined by means of HPLC-MS. Results are given in the following table11. TABLE 11 Microbubbles Emulsion DSPE-PEG Example D_(V50) (μm) D_(N)(μm) D_(V50) (μm) D_(N) (μm) (% w/w) 14a 2.6 2.3 1.9 1.1 0.0 14b 2.5 2.33.4 1.3 35.5 14c 2.5 2.3 2.2 1.2 37.9

As inferable from the above results, the subsequent addition of aDSPE-PEG suspension to the formed emulsion allows introducing relativelyhigh amounts of DSPE-PEG in the composition of the stabilizing layer (inthis case more than 30% of the total weight of the phospholipids formingthe stabilizing envelope), without negatively affecting the finalproperties of the microbubbles.

Similar results can be obtained with other PEG-modified phospholipids,in particular DSPE-PEG2000-Biotin or DSPE-PEG2000-Maleimide, and withpeptide bearing phospholipids, in particularDSPE-PEG2000-maleimide-SATA-RGD4C. This latter peptide bearingphospholipid can be prepared according to known techniques, by reactingthe RGD-4C peptide with SATA, deprotecting the thiol group of SATA andreacting the deprotected RGD4C-SATA with DSPE-PEG2000-maleimide. Thepreparation method described in “Development of EGF-conjugated liposomesfor targeted delivery of boronated DNA-binding agents”, by Bohl Kullberget al., Bioconjugate chemistry 2002, 13, 737-743, (describing theinsertion of a EGF protein in a DSPE-PEG-maleimide molecule), can beconveniently used.

Example 15

20 mg of a 80/20 (w/w) DSPC/DSPA mixture are dissolved in 1.6 ml ofcyclooctane at 80° C. and the suspension is added to 20 ml of distilledwater containing 10% (w/w) of PEG4000 (Fluka). The mixture is emulsifiedby using a high speed homogenizer (PolytronT3000) for 1 minute at 8000rpm.

DSPE-PEG1000 (0.29 μmole) and DSPE-PEG2000-maleimide-SATA-RGD4C (0.29μmole) are dissolved in EtOH/water 9/1 v/v; after evaporation, theobtained lipid film is dried overnight at 25° C. and 0.2 mBar andresuspended in 320 μl of water at 60° C.

The obtained solution is added to the emulsion previously prepared andthe resulting emulsion is heated under stirring at 80° C. for 1 hour,followed by cooling at to room temperature. The emulsion is thencentrifuged (1300 g/10 min) to eliminate the excess of phospholipid andthe floating microdroplets are recovered and resuspended in 40 ml ofPEG4000 10% solution.

The resulting emulsion is sampled in DIN8R vials (1 ml per vial) and thevials are frozen at −50° C. for 2 hours (Christ Epsilon lyophilizer),then freeze-dried at −25° C. and 0.2 mBar for 12 hours, with a finaldrying step at 30° C. and 0.05 mBar for 6 hours.

The lyophilized product is then exposed to an atmosphere containing 35%of perfluoro-n-butane and 65% of nitrogen and the vials are sealed.

The product is finally dispersed in a volume of water twice that of theinitial volume by gentle hand shaking.

Similar results are obtained when the DSPC/DSPA mixture is replaced by a80/20 DSPC/Stearic acid mixture.

Example 16

10 mg of a 1:1 (w/w) DPPS/DSPC mixture are added to about 10 ml of a 10%(w/w) mannitol aqueous solution.

The mixture is heated at 70° C. for 15 minutes and then cooled at roomtemperature (22° C.). Cyclooctane is added at a flow rate 0.2 mL/minthrough an inlet of a micromixer (standard slit Interdigidital microMixer, housing SS 316Ti with nickel-on-copper inlay, 40 μm×300 μm,Institut für Microtechnik Mainz GmbH) to the aqueous phase circulatingat 20 ml/min at room temperature, for a total amount of 7.4% (v/v) oforganic solvent. Upon completion of the addition of the organic solvent,the emulsion is recirculated in the micromixer for additional 20minutes.

The emulsion is then divided into five aliquots of 2 ml each and it isintroduced into five vials DIN8R. Four vials are sealed and heated for30 minutes at temperatures of 60, 80, 100 and 120° C., respectively, asindicated in table 12, while the fifth is not heated.

The emulsions are then cooled to room temperature and the content of thefive vials is subjected to lyophilization according to the followingprocedure. 1 ml of each emulsion is collected into a DIN8R vial andfrozen at −5° C.; the temperature is lowered to −45° C. during 1 hourand the emulsion is then freeze-dried at −25° C. and 0.2 mbar during 12hours (Telstar Lyobeta35 lyophilizer), with a final drying step at 30°C. and 0.2 mbar for 5 hours.

The lyophilized product is then exposed to an atmosphere containing 35%of perfluoro-n-butane and 65% of nitrogen and then dispersed in a volumeof water twice than the initial one by gentle hand shaking. Table 12shows the result of the characterization of the final suspension ofmicrobubbles. TABLE 12 Number of μbubbles Heating D_(V50) D_(N)D_(V50)/D_(N) per ml of emulsion no heating 10.45 1.63 6.41 5.34 × 10⁷ 60° C. 4.85 1.32 3.67 7.83 × 10⁷  80° C. 5.34 1.29 4.14 8.51 × 10⁷ 100°C. 6.96 1.66 4.19 4.92 × 10⁸ 120° C. 3.05 1.50 2.03 8.69 × 10⁸

From the above results, it appears that by subjecting the formedemulsion to a thermal treatment allows to narrow the size distributionof the final microbubble suspension, while also increasing the totalnumber of microbubbles. In particular, by increasing the heatingtemperature above 100° C., it is possible to obtain a relatively narrowsize distribution of microbubbles also in the absence of any washingstep of the emulsion, as well as an increase of the total number ofmicrobubbles in the suspension.

Example 17

20 mg of 80/20 (w/w) DSPC/DSPA phospholipid mixture are dissolved in 1.6ml of cyclooctane at 80° C. and the suspension is added to 20 ml ofdistilled water containing 10% (w/w) of PEG4000 (Fluka).

The mixture is emulsified by using a high speed homogenizer(PolytronT3000) for 1 minute at 8000 rpm.

The resulting emulsion is heated under stirring at 80° C. for 1 hourthen cooled to room temperature. Afterwards it is centrifuged (1300 rpmfor 10 min) to eliminate the excess of phospholipid and the floatingmicrodroplets are recovered and resuspended in 40 ml of PEG4000 10%solution.

The resulting emulsion is sampled in DIN8R vials (1 ml per vial) and thevials are frozen at −50° C. for 2 hours (Christ Epsilon lyophilizer),then freeze-dried at −25° C. and 0.2 mBar for 12 hours, with a finaldrying step at 30° C. and 0.05 mBar for 6 hours.

The lyophilized product is then exposed to an atmosphere containing 35%of perfluoro-n-butane and 65% of nitrogen and the vials are sealed.

The product is finally dispersed in a volume of water twice that of theinitial volume by gentle hand shaking.

Table 13 shows the characterization of of the emulsion and of themicrobubble suspension. TABLE 13 Emulsion Microbubbles D_(V50) (μm)D_(N) (μm) D_(V50) (μm) D_(N) (μm) D_(V50)/D_(N) <1.5 μm 2.89 1.66 2.511.27 1.98 22.1

Example 18

Distilled water (10 ml) containing 10 mg of DPPS and 1 g of mannitol isheated to 70° C. during 15 minutes then cooled to room temperature.DPPE-MPB(1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide] Na salt-Avanti Polar Lipids) is added (4.8% by weight-0.5mg). This phospholipid is dispersed in the aqueous phase using aultrasound bath (Branson 1210-3 minutes).

Perfluoroheptane (0.8 ml from Fluka) is emulsified in this aqueous phase(cooled with a ice bath) using a high speed homogenizer (Polytron®T3000, 15000 rpm, 1 minute).

The resulting emulsion showed a median diameter in volume (D_(V50)) of2.3 μm and a mean diameter in number (D_(N)) of 2.1 μm as determinedwith a Malvern Mastersizer.

The emulsion is washed twice by centrifugation then resuspended in 9.5ml of a 10% solution of mannitol in distilled water. The washed emulsionis frozen (−45° C., 5 minutes) then freeze dried (under 0.2 mBar, for 24hours).

Atmospheric pressure is restored by introducing a 35/65 mixture of C₄F₁₀and air. The lyophilisate is dispersed in distilled water (20 ml),microbubbles were washed once by centrifugation and then redispersed in4 ml of an EDTA containing phosphate buffered saline (molar composition:10 mM phosphate, 2.7 mM KCl, 137 mM NaCl, 10 mM EDTA), containing 3.4 mgof thioacetylated avidin, 400 μl of a hydroxylamine solution (13.92 mgin PBS 50 mM, pH: 7.5) were added to deprotect the thiol group of thethioacetylated avidin.

The suspension is stirred by inversion on a disk rotator (FisherScientific) for 2 hours. Then 150 μl of NaOH 1N were added.

The so obtained avidin-labelled microbubbles were washed twice with PBSby centrifugation (10000 rpm, 10 minutes, Sigma centrifuge 3K10). Themicrobubbles suspension obtained is analysed using a Coulter countershowing a D_(V50) diameter of 1.6 μm and a D_(N) of 1.2 μm.

The efficacy of targeted microbubbles composition was tested both invitro and in vivo.

In Vitro Experiment:

To test the effective bonding of acetylated avidin to the surface of themicrobubbles, two sets of fibrin containing wells were prepared. In thefirst set, only a fibrin surface is present. In the second set, thefibrin is pre-treated with a biotin-labelled antifibrin peptide (DX-278,disclosed in WO 02/055544). Microbubble suspensions prepared as abovewere added to the wells (5×10⁸ microbubbles/well). After 2 hours ofincubation (upside down) and several washings, the fibrin surfaces inthe two set of wells were observed by means of an optical microscope.While essentially no microbubble could be observed in the wells withoutthe biotinylated antifibrin peptide, a massive coverage of microbubblesweas observed in the biotinylated antifibrin peptide containing wells.

In Vivo Experiment:

A thrombus is formed in the abdominal aorta of two rabbits by the FeCl₃method (Lockyer et al., 1999, Journal of Cardiovascular Pharmacology,vol 33, pp 718-725).

Echo imaging is performed with an HDI 5000 ultrasound machine (Philips),pulse inversion mode, L7-4 probe, MI: 0.07.

A biotinylated antibody (CD41 specific for the GPIIB/IIIA receptor ofactivated platelets) is then injected intravenously to the two rabbit.

After 30 minutes, the microbubble suspension comprising avidin-labelledmicrobubbles is injected intravenously (1×10⁹ microbubbles/ml) in thefirst rabbit. Fifteen minutes after the injection, a strongopacification of the thrombus is observed for the suspension. Thisopacification is still visible after at least one hour from theinjection.

The same amount of the microbubble suspension without avidin-labelledmicrobubbles is injected intravenously in the second rabbit. Only alight opacification of the thrombus is observed.

1. An injectable aqueous suspension of microbubbles filled with abiocompatible gas and comprising a stabilizing layer predominantlycomprising a phospholipid, wherein at least 10% of the total volume ofgas contained in the microbubbles is contained in microbubbles with adiameter of 1.5 μm or less.
 2. The aqueous suspension according to claim1 wherein at least 25% of the total volume of gas contained in themicrobubbles is contained in microbubbles with a diameter of 1.5 μm. 3.The aqueous suspension according to claim 1 wherein at least 50% of thetotal volume of gas contained in the microbubbles is contained inmicrobubbles with a diameter of 1.5 μm
 4. The injectable suspensionaccording to claim 1 wherein at least 70% of the total volume of gascontained in the microbubbles is contained in microbubbles with adiameter of 1.5 μm
 5. The aqueous suspension according to claim 1wherein said microbubbles have a D_(V50)/D_(N) ratio of about 2.00 orlower.
 6. The aqueous suspension according to claim 1 wherein saidmicrobubbles have a D_(V50)/D_(N) ratio of about 1.80 or lower.
 7. Theaqueous suspension according to any one of the preceding claimsobtainable by a method which comprises the steps of: a) preparing anaqueous-organic emulsion comprising i) an aqueous medium includingwater, ii) an organic solvent substantially immiscible with water, iii)an emulsifying composition of amphiphilic materials comprising more than50% by weight of a phospholipid and iv) a lyoprotecting agent; b)lyophilizing said emulsified mixture, to obtain a lyophilized matrixcomprising said phospholipid; c) contacting said lyophilized matrix witha biocompatible gas; and reconstituting said lyophilized matrix bydissolving it in a physiologically acceptable aqueous carrier liquid. 8.A method for diagnostic imaging comprising administering to a subject acontrast-enhancing amount of an aqueous suspension of any one of claims1 to 6 and imaging at least a part of said subject.
 9. A method fordiagnostic imaging comprising administering to a subject acontrast-enhancing amount of an aqueous suspension of any one of claims1 to 6 and imaging at least a part of said subject wherein said imagingcomprises insonating said subject by means of an ultrasound devicegenerating an ultrasound wave with a predetermined transmit frequency,from which a corresponding resonance size of microbubbles is determined,and administering a contrast agent comprising gas-filled microbubbleshaving a narrow size distribution and a mean size close to half theresonance size.
 10. A method for preparing a lyophilized matrix which,upon contact with an aqueous carrier liquid and a gas, isreconstitutable into a suspension of gas-filled microbubbles stabilizedpredominantly by a phospholipid, said method comprising the steps of: a)preparing an aqueous-organic emulsion comprising i) an aqueous mediumincluding water, ii) an organic solvent substantially immiscible withwater comprising, dispersed therein, an emulsifying composition ofamphiphilic materials comprising more than 50% by weight of aphospholipid and iv) a lyoprotecting agent; b) lyophilizing saidemulsified mixture, to obtain a lyophilized matrix comprising saidphospholipid.
 11. The method according to claim 10 wherein thelyoprotecting agent is dissolved in the aqueous carrier.
 12. The methodaccording to claim 10 wherein the lyoprotecting agent ispolyethylenglycol.
 13. A method for preparing an injectable contrastagent comprising a liquid aqueous suspension of gas-filled microbubblesstabilized predominantly by a phospholipid, which comprises the stepsof: preparing a lyophilized matrix according to the method of any one ofthe preceding claims 10 to 12; contacting said lyophilized matrix with abiocompatible gas; and reconstituting said lyophilized matrix bydissolving it into a physiologically acceptable aqueous carrier liquid,to obtain a suspension of gas-filled microbubbles stabilizedpredominantly by said phospholipid.